We developed cloned populations from the commonly available, well-characterized cell line HUT-78. These cloned cells grow permanently after infection with isolates of human T-lymphotropic virus type III, also called lymphadenopathy virus (HTLV-III/LAV), from patients with acquired immune deficiency syndrome and related syndromes. In contrast, activated human T cells are lysed after HTLV-III/LAV infection. The infected cloned cells have been in culture continuously for 6 months and have produced high levels of extracellular reverse transcriptase (400,000 cpm/ml). This level is comparable to that of similarly infected normal human T cells. Three weeks after infection with HTLV-III/LAV, more than 90% of the cloned HUT-78 cells lysed; the remaining cells continued to grow. Approximately 80% of these cells expressed HTLV-III/LAV antigens by immunofluorescence. The extracellular virus of the chronically infected cell line was shown to be similar to other HTLV-III/LAV isolates by competition radioimmunoassay, by reactivity with human serum, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This HTLV-III/LAV-infected immortalized cell line enables the continuous production of large amounts of virus.