Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan. The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes. Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches. Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures. Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M). In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions. However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites. The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).