Gilthead seabreams were exposed to benzocaine, 4-aminobenzoic acid ethyl ester, 57 mg/l in sea water for 3 min, daily, for 2 or 3 consecutive days. The fish were killed 20 hr after the last treatment. Benzocaine treatment for 2 or 3 days resulted in 57% and 67% inhibition of liver microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities, respectively. The total cytochrome P450 content of fish liver microsomes was unaltered following the 2-day benzocaine treatment. However, additional 3 min benzocaine treatment on day 3 reduced cytochrome P450 level by 50%. Benzocaine produced type II difference spectra with rabbit liver microsomes. Difference spectra of fish liver microsomes elicited by benzocaine were complex. The position of peak and intensity were greatly influenced by the concentration of benzocaine.