The role of the NADPH oxidase homolog 1 (Nox1) in plasma membrane H(+) conductance and cellular H(+) production was investigated in 3T3 cells stably expressing Nox1 (Nox1 3T3) compared to vector-expressing control cells (mock 3T3). In whole cell patch clamp experiments both Nox1 and mock 3T3 expressed a similar H(+) conductance (Nox1 3T3, 13.2+/-8.6 pS/pF; mock 3T3, 16.6+/-13.4 pS/pF) with a number of similar characteristics (e.g., current-voltage relations, current activation kinetics, Zn(2+)-sensitivity). When the intracellular pH of cells was alkalinized with NH(4)Cl, rates of intracellular acidification were significantly higher in Nox1 3T3 compared to mock 3T3. Nox1 3T3 showed a time course of acidification that followed a double-exponential function with a fast and a slow component of, on average, tau=165 s and 1780 s, whereas mock 3T3 showed only a single slow tau of 1560 s. Expression of Nox1 also caused cells to acidify the extracellular medium at higher rates than control cells; Nox1 3T3 released 96+/-19 fmol h(-1)cell(-1) of acid equivalents compared to 19+/-12 fmol h(-1)cell(-1) in mock 3T3. These data show that expression of Nox1 results in a mechanism that has the capacity to rapidly acidify the cytosol and generate significant amounts of acid. No significant effect of Nox1 expression on the plasma membrane H(+) conductance was found.