The T-lymphoblastoid cell line CEM, persistently infected with herpes simplex virus type 1, has been used to examine the antiviral efficacy of human alpha interferon and acyclovir, both alone and in combination. Acyclovir and interferon each produced dose-dependent decreases in virus titer at concentration ranges of 1 to 100 microM (approximately 0.225 to 22.5 micrograms/ml) and 10 to 10,000 U/Ml, respectively. Mean reductions in titer of 1.9 and 4.2 log10 PFU/ml were observed with 100 microM acyclovir and 10,000 U of interferon per ml, respectively, on day 10 of treatment. The combination of 100 microM acyclovir and 10,000 U of interferon per ml produced the most rapid fall in virus titer of all regimens examined and elimination of infections virus by day 7. Prolonged treatment (greater than 10 days) with acyclovir or alpha interferon was accompanied by a gradual return of virus titer to control levels despite the continuous presence of drug. Virus preparations isolated from such cultures were tested for antiviral agent sensitivity by a plaque reduction method. Acyclovir-exposed isolates were found to be acyclovir resistant, with 50% inhibitory doses of greater than 200 microM, and to be thymidine kinase deficient. Alpha interferon-exposed isolates were not interferon resistant. These results suggest that, in persistently herpes simplex virus-infected CEM cells: (i) combination treatment with 100 microM acyclovir and 10,000 U of alpha interferon per ml is more effective in reducing virus titer than either agent alone; (ii) prolonged exposure to drug may result in development of resistance by either the virus strain or the host cell system; and (iii) development of acyclovir resistance by herpes simplex virus in lymphoid cells is mediated by thymidine kinase.