The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 microg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 x 10(-4) +/- 2.5 x 10(-4) (mean +/- standard error), and that of HSV type 2 isolates was 15.0 x 10(-4) +/- 4.6 x 10(-4). The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 x 10(-4) to 15.0 x 10(-4) among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.