The potentiation of the antiviral activity of acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] by polyene macrolide antibiotics has been studied as a function of the macrolide structure. The 12 polyenes chosen for this study represented the major structural groups of these antibiotics and induced in mammalian cells repairable membrane alterations or irreversible cell damage. The potentiating activity of the polyene macrolides was determined based on the differential decrease of in vitro production of infectious virions in the presence of acyclovir alone or in combination with the polyene. Pseudorabies virus, a representative herpesvirus susceptible to acyclovir, was replicated in BHK-21 cells grown in serum-free medium to avoid the interference of serum factors in the polyene macrolide-cell interaction. The potentiation activity of the polyene antibiotics was concentration dependent. The enhancement of the antiviral activity of acyclovir was observed at polyene concentrations which had no direct effect on pseudorabies virus replication in BHK-21 cells. The optimal potentiating concentrations of polyenes were 2 to 15 times lower than that inducing 50% of potassium efflux from BHK-21 cells. The highest potentiating activity was observed for the methyl ester of the trimethylammonium derivative of aureofacin B, which reduced the pseudorabies virus titer by two orders of magnitude. Potentiation by polyene macrolides appeared to coincide with the K+-dependent membrane repair process. The acyclovir potentiating activity was associated with polyene macrolide antibiotics having a large and rigid macrolide ring (amphotericin B and aureofacin). Polyene antibiotics with small and rigid (pimaricin and filipin) or large but flexible (nystatin A1 and lienomycin) macrolide rings showed no potentiation of the antiviral effect of acyclovir.