1. We compared the intrinsic clearance (CL(int)) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors. 2. CL(int) values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9 +/- 3.4, 18 +/- 7.2, 5.1 +/- 4.9, 6.3 +/- 3.3, 9.8 +/- 5.8 and 22 +/- 14 microl min(-1)/10(6) cells, respectively, and they correlated well with corresponding CL(int) values using cryopreserved hepatocytes from 25 different donors. 3. CL(int) values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL(int) in fresh and cryopreserved hepatocytes for each of the three livers (p < 0.002) and the geometric mean of the ratio of fresh to cryopreserved CL(int) values was 1.03. 4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.