A new isolating procedure of human hepatocytes has been developed using two-step collagenase digestion by a non-perfusion procedure (NP) of non-wedge liver biopsies. 1. A yield of 2-7 x 10(6) hepatocytes/g liver, 52-95% viability and 13-75% attachment were obtained from liver biopsies weighing 6-60 g, comparable to that obtained when using the classical perfusion procedure (P) to isolate human hepatocytes from wedge liver samples of 50-150 g. 2. In culture, human hepatocytes obtained by NP remained attached to plastic for up to 5 days and displayed the usual morphological characteristics. Their metabolic capacities, assessed by liver-specific albumin and urea synthesis and by CYP-dependent and conjugation pathways, were equivalent to those of human hepatocytes obtained by P. In addition, they responded adequately to specific CYP inducers, demonstrating that they constitute a model in which human drug metabolism and toxicity studies can be performed.