Experimental conditions affecting the successful propagation of HIV-1 from the plasma of seropositive individuals were examined. It was determined that whole blood samples collected with lithium heparin as the anticoagulant, immediate plasma separation, and immediate culturing were best suited for obtaining viable virus from plasma. Virus was isolated by infecting fresh phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cells (PBMCs) with plasma followed by weekly cocultivation with new target cells. The plasma virus isolation rate was the greatest and HIV-1 titers were the highest for those individuals with less than 200 CD4+ cells/mm3 and decreased as the level of CD4+ cells approached normal values. We were able to obtain positive cultures from 29.5% of those patients with CD4+ counts greater than 500/mm3. HIV-1 titers in plasma also correlated with high serum p24 antigen levels when serum was treated with glycine to dissociate antigen-antibody complexes.