The metabolism of aquaculture antibiotics on the piscine, hepatic cytochrome P-450 (CYP) system has not yet been defined. Fifty summer flounder, maintained at 20°C, were fed ormetoprim-sulfadimethoxine (Romet-30®) at 1% body weight daily and were randomly sampled before treatment and on days 1, 6, 10 and 21 after treatment. Western blotting of hepatic microsomes included goat antirat CYP1A1 and rabbit antihuman CYP3A4 serum. Catalytic activities comprised: 3-cyano-7-ethoxycoumarin (CEC), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), resorufin benzyl ether (BzRes). Treatment induced CYP1A1 and CYP3A4 expression. Dealkylation of CEC (CYP1A2) was increased after treatment. Romet-30 inhibited CYP3A4 activity measured by BFC, but induced BzRes CYP3A4. The usefulness of mammalian antibodies for piscine P-450 Western blotting was demonstrated. The hepatic P-450 1A2 and 3A4 metabolism was quantifiable by kits developed for mammalian microsomes.