1. The effects of phenobarbital (PB), beta-naphthoflavone (beta-NF), omeprazole (Omep) and rifampicin (Rif) on drug-metabolizing activities in dog hepatocytes, cultured with William's medium E, were examined. 2. The drug metabolizing activities of the hepatocytes decreased during culture; 7-ethoxycoumarin O-deethylase (ECOD) activity was nearly 70% of initial value at 72 h, but 7-methoxycoumarin O-demethylase (MCOD), 7-propoxycoumarin O-depropylase (PCOD), progesterone 6 beta-hydroxylase (6 beta-OH-P), progesterone 16 alpha-hydroxylase (16 alpha-OH-P), progesterone 21-hydroxylase (21-OH-P), 7-ethoxyresorufin O-deethylase (EROD) activities and total cytochrome P450 content were approx. 50%. 3. When the hepatocytes were cultured with PB, the enzyme activities increased time- and dose-dependently. MCOD, ECOD and PCOD activities increased 5-8 fold with 2 mM PB in 96 h. Similar results were obtained for 6 beta-OH-P, 16 alpha-OH-P and 21-OH-P activities, and total cytochrome P450. The effect of PB was abolished when 2.5 microM cycloheximide or 0.1 microM actinomycin D was included in the culture. 4. Treatment of hepatocytes with 40 microM beta-NF for 72 h resulted in 25-fold elevation of EROD activity. beta-NF enhanced PCOD activity approx. six-fold, while ECOD increased only slightly, and 7-MCOD negligibly. 5. Omep (100 microM) increased EROD activity nearly 10-fold, and 25 microM Rif increased 6 beta-OH-P activity approx. 8-fold, but ECOD only slightly. 6. Western blot analysis of microsomes from cultured dog hepatocytes with anti-rat CYP 2B1 antibodies indicated that PB increased an immunochemically-reactive protein. The protein showed the same mobility as the major dog P450 isozyme (cytochrome P450 PBD-2 or CYP 2B11) purified from liver microsomes of PB-treated male beagle dog. In a similar manner, induction of cytochrome P450 PBD-1 (CYP 3A12) by PB was confirmed.