To determine the most convenient and least expensive regimen for prevention of recurrent herpes simplex virus (HSV) infection after marrow transplantation, we conducted a randomized, double-blind comparison of intravenous acyclovir 250 mg/m2 and placebo given once daily for four weeks. Six of 14 acyclovir and nine of 13 placebo recipients shed HSV during prophylaxis. All nine culture-positive placebo recipients developed associated lesions during prophylaxis compared to four of six acyclovir recipients. Median time to first culture-positive lesion was significantly delayed by acyclovir compared to placebo (33 days after transplant vs. 10; P = 0.05). Acyclovir-resistant HSV was recovered from one acyclovir recipient while receiving prophylactic acyclovir, and from two placebo recipients during subsequent administration of therapeutic acyclovir. Once-daily intravenous acyclovir can significantly delay time to appearance of culture-positive HSV lesions after marrow transplant, but virological and clinical breakthrough may occur and optimal prevention will require administration of intravenous acyclovir more than once daily.