Quantitation of human immunodeficiency virus type 1 (HIV-1) in plasma is frequently used to monitor virus load and assess the effect of antiviral agents, but information regarding the stability of the virus is limited. Plasma from patients with CD4 cell counts of < 300/microL was tested by quantitative cultures 1, 4, and 24 h after phlebotomy and after storage at -75 degrees C. Viremia was detected in 18 (69%) of 26 patients. Of 16 samples, 2 (13%) at 4 h and 6 (38%) at 24 h had a significantly lower titer than the sample cultured at 1 h. No culture result after the freezing step was significantly different from the 1-h reference value. The decrease in virus titer over time was observed primarily in patients with CD4 cell counts of > 100/microL. Plasma should not be stored for > 2-4 h at room temperature for HIV-1 culture. If immediate processing is not an option, plasma can be frozen shortly after phlebotomy for later testing without a significant loss of infectious virus.