For many years, our group has been working on the development of a novel approach for prediction of topical antiviral efficacies of acyclovir (ACV) formulations in the treatment of cutaneous HSV-1 infections in hairless mice. This approach is based on estimation of the free drug concentration (C * ), at the target site using the experimentally determined in vitro fluxes (J). First, a relationship between ACV flux (using controlled delivery transdermal patches) and efficacy was established by applying a novel method for evaluation of topical and systemic efficacies in hairless mice cutaneously infected with HSV-1 [Gonsho et al., Int. J. Pharm., 65 (1990) 183-194]. Then C * for 50% antiviral efficacy (C * 5 0 ) was determined from the plasma levels of ACV that inhibited cutaneous lesion formation in 50% of the animals treated systemically [Imanidis et al., Pharm. Res., 11 (1994) 1035-1041]. Using this C * 5 0 along with the ACV flux resulting in 50% topical efficacy (J 5 0 ), the in vivo dermis permeability coefficient (P D ) for ACV was estimated using the equation: P D = J 5 0 C * 5 0 . We can then predict efficacy of a topical formulation by calculating C * achieved by the formulation from the experimentally measured flux (J) data using the equation: C * = JP D [Lee et al., Int. J. Pharm., 93 (1993) 139-152]. Based on this analysis, it is clear that the formulation which produces a higher skin flux can achieve a higher C * , resulting in a higher predicted efficacy. To further investigate the significance of skin flux, an extensive examination of the C * concept was undertaken using several ACV formulations. For determination of C * , the in vitro skin fluxes were measured in an in vivo-in vitro experimental design that approximated the in vivo antiviral treatment protocol. Then in vivo efficacies were measured using the hairless mouse model for cutaneous HSV-1 infections. Our results indicate that over a wide range of efficacies, the predictions based on C * (estimated from the in vitro experimental fluxes) are in good agreement with the in vivo efficacies. These findings support the validity of the C * concept and underscore the significance of high skin fluxes produced by topical formulations for achievement of therapeutic levels of ACV at the target site.