Hydroxylation and acetylation of sulphadimidine (SDD) and the deacetylation of N4-acetyl SDD was investigated in cultured hepatocytes from male and female rats, from male and female goats and from female sheep and cattle. Significant sex differences were observed for hydroxylation of SDD in hepatocytes from rat and goat. In goat, sheep and cow hepatocytes, the hydroxylation pathway is relatively important, whereas in rat hepatocytes, acetylation is predominant. Hepatocytes of all four species deacetylated N4-acetyl SDD. In ruminant hepatocytes, deacetylating activity was of considerable importance, whereas in rat hepatocytes, it appeared a minor pathway of metabolism. Similar to the in vivo situation, formation of N4-acetyl SDD in cultured hepatocytes results from an equilibrium of acetylation and deacetylation. A good correlation was found between results in isolated hepatocytes and previous findings in vivo, both in levels of species-related activities and in acetylation-hydroxylation ratios. In conclusion, cultured hepatocytes appear a useful in vitro model to study comparative sulfonamide metabolism.