Biotransformation of caffeine and theophylline and the effect of two well-known inducers of P-450 isozymes, namely phenobarbital (PB) and methylcholanthrene (3-MC) were studied in cultured hepatocytes from six human adult donors. Hepatocytes co-cultured with rat liver epithelial cells maintained a higher metabolic capacity than pure cultures. PB treatment of cultured hepatocytes for 3 days slightly increased the rate of caffeine metabolism 1.4 +/- 0.5-fold (N = 6) vs controls, and theophylline metabolism 1.2 +/- 0.4-fold (N = 6), whereas 3-MC treatment increased metabolism markedly 5.8 +/- 2.3- and 3.3 +/- 1.1-fold (N = 6) vs controls for caffeine and theophylline, respectively. Paraxanthine and theophylline formations from caffeine were the most induced by 3-MC. Their increase was significantly correlated (rs = 0.89, P less than 0.007) but not with TB formation, suggesting that at least two isozymes of the P-450IA family are involved in the first demethylations of caffeine. In addition, the N-1 demethylation of theophylline (mean increase of 554% vs controls) was not correlated with the N-1 demethylation of caffeine (mean to increase 247% vs controls) for the same donor after 3-MC treatment, suggesting that these two demethylations are mediated by a different P-450.