Human livers were used in investigations of mexiletine biotransformation in vitro. The major metabolic pathways of mexiletine oxidation, to form hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM), were characterized in liver cell preparations. The localization of reactions in the microsomal fraction, their heat lability, NADPH requirement and inhibition by prototype cytochrome P-450 (P-450) inhibitors (CO, SKF 525-A, metyrapone and quinidine) implied that they were catalyzed by P-450. Kinetic studies of reactions were performed in microsomes from five different livers. Eadie-Hofstee plots of data gave no indication of systematic deviation from linearity, suggesting that over the range of mexiletine concentrations examined (3.3-133.3 microM), HMM and PHM were formed by a single enzymatic site. Within a liver preparation, Km and Vmax values for HMM and PHM formation were similar. Between livers, Km values of reactions were similar with only a 1.8-fold range for each reaction, whereas Vmax values showed 7.2- and 7.8-fold ranges for HMM and PHM production, respectively. There was a very strong correlation between Vmax values for both reactions. These results, coupled with a parallel effect of inhibitors (SKF-525A, metyrapone, alpha-naphtoflavone and quinidine) on HMM and PHM formation, argue that both reactions are mediated by a common P-450 or closely related isozymes. In addition, the present in vitro results support the hypothesis that the genetically variable P-450 db 1 isozyme catalyzes the oxidation of mexiletine.