The first steps in the metabolism of caffeine and chlorzoxazone are primarily catalysed by CYP1A2 and CYP2E1, respectively. Accordingly, these compounds have been developed as metabolic probes for non-invasive phenotyping of these two P450s. Their specificities, however, have been shown to overlap. In this study, 140 mg of caffeine and 500 mg of chlorzoxazone were administered alone or together in 16 healthy subjects under standardized conditions. The metabolites of these two probes were measured in the blood and also in the urine for caffeine. CYP1A2 activity was determined either by the paraxanthine/caffeine ratio in the blood or by the usual caffeine metabolic ratio in the urine. The CYP2E1 activity was determined by the 6-OH-chlorzoxazone/chlorzoxazone ratio in blood. CYP1A2 activities measured in blood and urine were highly significantly correlated. CYP2E1 activity was not modified when chlorzoxazone was given together with caffeine. In contrast, an inhibition of CYP1A2 by chlorzoxazone was demonstrated by a 16% decrease in the caffeine metabolic ratio in urine when both caffeine and chlorzoxazone were given together. Under the same conditions, the paraxanthine/caffeine ratio in plasma also decreased by about 20%. These results were confirmed in vitro by the incubation of 1 mM caffeine with human hepatic liver microsomes in the presence of 0.4 mM chlorzoxazone. The overall metabolism of caffeine decreased by 38% compared to controls incubated without chlorzoxazone.(ABSTRACT TRUNCATED AT 250 WORDS)