The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique (SIET) was applied to measure H(+) and NH4(+) fluxes in specific cells on the skin of zebrafish larvae. NH4(+) extrusion was relatively high in H(+) pump-rich cells (HRCs), which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH4(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH4(+) gradients at the larval surface. Moreover, coupled decreases in NH4(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligoneculeotides also decreased NH4(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion. Key words: zebrafish, ammonia, H+-ATPase, ionocytes.