This report describes a donor in a therapeutic donor insemination program who asymptomatically acquired a primary herpes simplex virus type 2 (HSV-2) infection from his long-standing sexual partner. His fresh semen was used to inseminate two HSV-seronegative recipients; in one a primary HSV-2 infection developed, and in one it did not. Direct evidence of transmission from donor to recipient was documented by restriction enzyme analysis of the HSV-2 isolates obtained from the donor's semen and from the recipient's cervix. Because of the possibility of asymptomatic acquisition and transmission of HSV-2, semen donors and their sexual partners should undergo serologic screening for genital herpes using new, type-specific HSV serologic techniques.