Mangrove killifish, Rivulus marmoratus, are tolerant of prolonged periods of air exposure (>30 days). Air-exposed R. marmoratuseliminate more than 40% of their total ammonia through NH3volatilization; however, the sites and mechanisms are unclear. We hypothesized that the cutaneous surface is an important site of NH3volatilization in air-exposed R. marmoratus. Ion-selective microelectrodes were used to measure the NH4+concentration and pH in the boundary layer on the cutaneous surface of fish in water or air (acute: 1 h, chronic: 11 days). Following acute and chronic air exposure, there was a ∼18-fold increase in the NH4+concentration and a 0.3–0.6 pH unit increase on the cutaneous surface of R. marmoratus. In air-exposed fish, the calculated cutaneous partial pressure (PNH3) was 608–1251 μTorr,representing a 33- to 75-fold increase over control (immersed) fish. The PNH3 on the cutaneous surface water film was more than sufficient to account for the rate of NH3 volatilization under terrestrial conditions. Together, these data indicate that during air exposure, R. marmoratus utilize the cutaneous surface as a key site of NH3 volatilization.