The effectiveness of prodrug-enhancer combination in skin penetration enhancement was studied using acyclovir and its lipophilic prodrug, acyclovir butyrate, with octanol/water partition coefficient of 0.0123 and 0.402, respectively. In the in vitro diffusion experiment with rat skin, the total amount of acyclovir appearing in the receptor phase after administration of the aqueous suspension of acyclovir butyrate was smaller than that obtained after administration of acyclovir, but their permeability coefficients were almost equal. An enhancer, 1-geranylazacycloheptan-2-one (GACH) did not show a large penetration enhancement of acyclovir (3.37-fold) but demonstrated extensive enhancement effect on the prodrug (12.3-fold). Most of the prodrug appeared in the form of acyclovir in the receptor phase without GACH, but the appearance ratio of acyclovir to total flux decreased with an increase in pretreatment doses of GACH.