Nonionic diffusion and diffusion equilibrium of ammonia have been generally accepted as the mechanism of urinary ammonium excretion. However, these characteristics have not been examined directly in vitro. In the present studies, nonionic diffusion and diffusion equilibrium of ammonia were examined in rabbit cortical collecting tubules perfused in vitro. Collected fluid ammonium and pH were measured in tubules exposed to chemical gradients of NH3/NH+4. In tubules perfused with an acid perfusate free of ammonia and bathed with solutions containing NH4Cl, collected fluid ammonia failed to equilibrate across the epithelium except at slow flow rates. The estimated apparent permeability coefficient to NH3 was approximately 5 X 10(-3) cm/s. Predominant nonionic diffusion of NH3, rather than transport of NH+4, was indicated by alkalinization of luminal fluid in tubules exposed to peritubular NH4Cl and by the relative influence of peritubular NH+4 and NH3 on ammonia entry. In tubules perfused with an acid solution containing NH4Cl, little loss of ammonium was detectable, indicating a low permeability to NH+4. In contrast to the restricted diffusion of NH3 in cortical collecting tubules, proximal convoluted tubules exhibited a much higher apparent permeability to NH3. In conclusion, nonionic diffusion of NH3 accounted for most ammonium transport in the proximal convoluted tubule and in the cortical collecting tubule. However, there was relatively restricted diffusion in the collecting tubules; this may account for the failure of whole kidney ammonium excretion to obey quantitatively the predictions of nonionic diffusion and diffusion equilibrium of ammonia.