The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.