As part of a double-blind placebo-controlled study of the effect of thalidomide on body weight and the viral load of human immunodeficiency virus-seropositive patients, plasma and semen samples were analyzed for the presence of thalidomide. Patients were orally dosed with 100 mg of thalidomide/day for 8 weeks. Blood samples were obtained at baseline and weeks 4, 8, and 12, and semen was obtained at baseline and weeks 4 and 8. Samples were extracted with solid-phase cartridges and analyzed by liquid chromatography/tandem mass spectrometry using atmospheric pressure chemical ionization in the negative ion mode. Two of four patients taking thalidomide were able to provide semen samples. Both had detectable levels of thalidomide in their plasma (10-350 ng/ml) and semen (10-250 ng/g) at weeks 4 and 8. There was an apparent correlation between plasma and semen levels. Semen levels could be significantly greater for therapeutic doses of more than 100 mg/day. Since the threshold dose for birth defects and thalidomide exposure is not known, male patients are advised to use barrier contraception.