We showed here that the catalytic subunits BcNoxA and BcNoxB in B. cinerea both localize to the nuclear envelope, the ER and at times to the plasma membrane, whereat BcNoxA showed more concentrated localization around the nuclei, while BcNoxB localization was more dominant within the ER. Since Nox are transmembrane proteins postulated to be located in the outer membrane and to produce extracellular ROS, an ER localization of the complex was not assumed. However, until now there is no clear evidence for extracellular ROS derived from Nox. During pathogenic development extracellular ROS could either be used directly to attack the host, or they could serve as messenger molecules that are perceived to activate internal signaling processes. Interestingly, in the bcnox deletion mutants decreased ROS levels were neither detected in axenic culture by DAB staining or use of Amplex Red nor in planta using NBT staining. In fact for DbcnoxA and Dbcpls1 even a moderate enhancement was detected, which was also previously reported for other fungi. The observed reduction of H2O2 production after DPI treatment substantiates the unspecific effect of this inhibitor and supports the now generally accepted idea that secreted ROS in fungi is not produced by Nox but by alternative flavoenzymes. Hence, in B. cinerea a contribution of the Nox to extracellular ROS production is doubtful, and accordingly, the Nox might localize to a compartment other than the outer membrane. Even though we show a localization of BcNoxA and possibly BcNoxB in the ER, both proteins do not possess any ER retention signals. However, retention of a PI-103 PI3K inhibitor protein in the ER can result from various factors like the lack of a sorting signal, the presence of a signal for retrieval from pre-Golgi compartments or properties of the transmembrane domain. The exact mechanism of ER retention remains to be elucidated. Integral predictions of protein locations were done resulting in a prediction of all analyzed Nox proteins at the plasma membrane, except Nox2 from T. reseei which is predicted at the ER. The fact that the H. sapiens Nox1, 2 and 4 have repeatedly been shown to localize and function at the ER shows that software based localization predictions may differ from real localization. Recently, the yeast Nox Yno1 was also shown to be present in the ER like mammalian Nox1, Nox2 and Nox4. Besides a primary localization in the ER, Nox4 was reported to translocate to the plasma membrane when complexed with the stabilizing component p22phox, revealing that localization and function of a protein might not be restricted to a specific compartment or that proteins can be located in the ER for a period of time until they are needed and released. For example the chitin synthase 2 is expressed in the metaphase and WZ8040 EGFR/HER2 inhibitor retained in the ER through phosphorylation until further mechanisms stop this phosphorylation and release Chs2 from the ER.. According to these findings it is difficult to decide whether the ER is the final location of the catalytic BcNox subunits, or whether they are stored and modified there in order to be translocated. The fact that the GFP-BcNoxA fusion construct complemented the phenotype of the deletion mutant proves the functionality of the construct and supports a correct localization. However, the function of BcNoxA and BcNoxB within the ER is still unclear.