Incubation of opossum kidney clone P (OKP) cells in acid media (pH 6. 8) causes activation of Na(+)/H(+) exchanger 3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but was unaffected at 3-12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and 24 h, mirroring the increase in activity. Acid incubation caused a 76% increase in exocytic insertion of NHE3 into the apical membrane but had no effect on endocytic internalization at 6 h. Latrunculin B, an inhibitor of microfilament organization, inhibited the acid-induced increases in apical membrane NHE3, exocytic insertion of NHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at 6 h, there is an increase in apical membrane NHE3 that is due to stimulated exocytic insertion and is required for increased NHE3 activity. At 24 h, there is an additional increase in total cellular NHE3.