The effect of dose and enzymatic inhibition on the percutaneous absorption and metabolism of benzocaine was studied in vitro in the hairless guinea pig. At the dose level of 2 micrograms/cm2, benzocaine was rapidly absorbed and extensively metabolized (80%) by acetyltransferase. As the applied dose of benzocaine was increased to 40 and 200 micrograms/cm2, N-acetylation of benzocaine decreased to 44 and 34%, respectively, suggesting saturation of the acetyltransferase system. Total 14C absorption after benzocaine application was not significantly different between control and enzyme-inhibited skin and therefore does not appear to be affected by the extent of benzocaine metabolism during percutaneous penetration. Skin provides a significant first-pass metabolic effect for therapeutic doses of percutaneously absorbed benzocaine, and the primary metabolite formed, acetylbenzocaine, is biologically active.