End-product inhibition was explored as a mechanism for the lower clearance determination obtained from microsomes compared with hepatocytes. Triazolam, diazepam and phenytoin microsomal substrate depletion was reduced by 23, 34 and 39%, respectively, when incubated with their primary metabolites. Ki values of 28+/-6 and 11+/-1 microM were obtained when 4'-hydroxydiazepam and p-hydroxyphenytoin where incubated with diazepam and phenytoin, respectively. Alamethicin (a glucuronidation activator) was unsuccessful in alleviating these effects. IC50 values of 17, 32 and 18 microM for phenytoin and 83, 110 and 97 microM for diazepam were observed with salicylamide- (a glucuronidation inhibitor) treated hepatocytes, control hepatocytes and microsomes, respectively, when incubated with their primary metabolites. These differences suggest that metabolite concentrations in the vicinity of the enzyme are lower in hepatocytes compared with microsomes, reducing the likelihood of end-product inhibition in the former system. In conclusion, end-product inhibition may be more prominent in microsomes (in particular for substrate depletion assays where metabolism tends to be more extensive); results suggest that this phenomenon may contribute to the observed variations in metabolism characteristics and intrinsic clearance (CLint) between hepatocytes and microsomes.