The possibility was investigated of using frozen stocks of phytohemagglutinin (PHA)-stimulated normal human peripheral blood lymphocytes (PBL) in cocultivation with human immunodeficiency virus (HIV)-infected lymphocytes for the isolation of HIV. Fresh and cryopreserved PBL from eight healthy volunteers were compared for their susceptibility to HIV infection in vitro. Fresh lymphocytes, as well as lymphocytes that were stimulated with PHA before or after cryopreservation, displayed comparable susceptibilities to HIV infection in vitro. In addition, HIV was recovered in all cases when lymphocytes stimulated with PHA before or after cryopreservation were cocultured in parallel with PBL from 15 patients with acquired immune deficiency syndrome. However, the cryopreserved PBL were less efficient in isolating HIV from asymptomatic men.