The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL-6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process.