Subjects received topical penciclovir for 4 days during successive episodes of recurrent herpes labialis. Isolation of herpes simplex virus (HSV) was attempted from lesions obtained before initiation of treatment and on each day of therapy. Isolates remained sensitive to penciclovir when tested by a plaque reduction assay, and there was no significant change in sensitivity during any treatment course or between successive treatments. The proportion of nucleoside-resistant variants present within a subset of these isolates was further investigated using a more-sensitive plating efficiency assay. Although the proportion of antiviral-resistant HSV variants increased on successive days, it invariably remained a minor subpopulation. Moreover, isolates from successive episodes obtained before treatment showed no change in the proportion of resistant HSV variants. We conclude that antiviral-resistant variants, which are readily detected in HSV isolates from peripheral lesions, do not accumulate in the sensory ganglia of immunocompetent patients receiving multiple courses of nucleoside analogues.