Recurrent mucocutaneous lesions occur in many infants after completion of antiviral therapy of neonatal herpes simplex virus (HSV) infection. To determine whether these recurrences were caused by viruses that had become resistant to acyclovir or vidarabine, we tested the antiviral susceptibilities of 22 pretherapy and 32 posttherapy HSV isolates from 22 infants younger than 3 months of age. Sixteen had been treated with acyclovir and six with vidarabine. Antiviral susceptibilities were measured by an enzyme-linked immunosorbent assay and are expressed as the 50% inhibitory dose. All HSV isolates had a 50% inhibitory dose for acyclovir of less than 1.0 micrograms/ml. The mean vidarabine 50% inhibitory dose was 11.4 micrograms/ml for pretherapy isolates and 8.9 micrograms/ml for posttherapy isolates. Antiviral therapy did not select for recurrences with HSV resistant to acyclovir or vidarabine.