1. Predictions of in vivo intrinsic clearance from cryopreserved human hepatocytes may be systematically low. In the current study, the metabolite kinetics of a series of CYP3A4 substrates (benzodiazepines) in fresh human hepatocytes from five donors, via a major UK supplier, were investigated and compared with those previously reported (by the authors' laboratory) for cryopreserved human hepatocytes and hepatic microsomes. 2. A high incidence of autoactivation (up to tenfold) and heteroactivation (by testosterone, up to 14-fold) among the major pathways was observed. CYP capacity (V(max)) was marginally lower and 'affinity' constants (K(M), S(50)) were marginally greater compared with cryopreserved hepatocytes. 3. Average intrinsic clearance (based on maximal clearance, CL(max)) was sevenfold lower than in cryopreserved hepatocytes (reflecting sensitivity of intrinsic clearance estimation in vitro to mechanistic parameter values, particularly those involving atypical kinetics), but scaled intrinsic clearances for fresh (and cryopreserved) hepatocytes were within the range previously determined in hepatic microsomes. 4. There was no evidence from this series of studies that fresh hepatocytes provide quantitatively improved estimates of intrinsic clearance over cryopreserved hepatocytes.