We have reported previously that gp91phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91phox failed to exhibit outward proton currents, and concluded that gp91phox does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C-91, in which gp91phox is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C-91 was observed in the plasma membrane and intracellular membranes of 30% of the transfected COS-7 cells. In the remaining COS-7 cells, EGFP-C-91 was detected in the intracellular membranes only. In CHO cells EGFP-C-91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-cell configuration, outward currents were recorded from COS-7 cells expressing gp91phox. These increased in magnitude and lost their 'droop' over time as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by approximately 60 mV for a 1 unit difference in bath pH. Zn2+ inhibited the outward currents observed in COS-7 cells expressing gp91phox. The tail current reversal potential was -64 mV at a pH(o) (external pH) of 8.0, -40 mV at pH(o) 7.4 and -8 mV at pH(o) 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91phox. Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91phox. The presence of outward proton currents in COS-7 cells expressing gp91phox provides further support for our proposed role for gp91phox as the NADPH oxidase-associated proton channel.