The present study was performed to clarify the effect of H89, an inhibitor of cAMP-activated protein kinase (protein kinase A; PKA), on Na(+) absorption in fetal rat alveolar type II epithelium. H89 stimulated the Na(+) absorption by increasing the open probability (Po) and number of a nonselective cation (NSC) channel composed of four alpha subunits of epithelial Na(+) channel (ENaC). Brefeldin A (BFA), an inhibitor of intracellular protein translocation, blocked the stimulatory action of H89 on the Na(+) absorption by interrupting the action of H89 on the Po and number of the NSC channel. H85, an inactive form of H89, showed an effect similar to H89, suggesting that H89 does not show its effect by inhibiting PKA, but acts on the channel depending the structure. These observations indicate that: (1) the H89 induced increase in number of the channel at the apical membrane is due to translocation of alpha subunit of ENaC to the apical membrane, (2) the elevation of Po of the channel is mediated through translocation of a protein activating alpha subunit of ENaC, and (3) the effect of H89 is dependent on its structure without any relation to PKA.