Tissue concentration of nonesterified fatty acids (NEFA) exerting an inhibitory effect on renal N-acetyltransferase activity was determined in the isolated perfused rat kidney. Concurrently, O₂ consumption and the N-acetylation rate of p-aminohippurate (PAH) were measured. In a second series of experiments, the mode of inhibition of NEFA on acetylating enzyme(s) was studied. Amount and pattern of NEFA as well as N-acetylation rate of PAH in the kidney were related to the O₂ consumption: lower O₂ supply corresponded to a higher tissue NEFA concentration as well as lower N-acetylation rate, increased O₂ supply resulted in a low tissue NEFA concentration and an increased N-acetylation rate of PAH. Decreasing O₂ supply elevated the tissue concentration of linoleate especially. NEFA with a carbon chain length of C₁₆-C₂₀ inhibited renal N-acetyltransferase activity in vitro competitively according to the sequence C₁₆, C18, C_18:1, C_18:2, C_18:3=C₂₀. It is inferred that hypoxia interferes with the N-acetylation of PAH in the rat kidney by increasing the content and changing the pattern of fatty acids, thereby inhibiting the N-acetylating enzyme(s).