Early events in the infection of the human T-lymphotropic virus type-I (HTLV-I)-positive MT-4 cell line by the acquired immune deficiency syndrome (AIDS) retrovirus HTLV-III were investigated. The virus was adsorbed completely to the cells within 60 min incubation after inoculation of the virus. Then, infected MT-4 cells started to produce the HTLV-III-specific antigens between 12 and 24 hr postinfection. Synthesis of the viral antigens consisting of 120K, 46K, 24K, and 17K polypeptides was suppressed by the treatment of the virus-infected MT-4 cells with cytosine arabinoside (Ara-C) or by the treatment of the virus with anti-HTLV-III-positive sera. The progeny of the virus released from the infected MT-4 cells was titrated by a newly developed plaque-forming assay method and reverse transcriptase activity. The maximum activity of HTLV-III (3 X 10(5) PFU/ml) was observed on Days 4 and 5 p.i. Most of the viral activities in this preparation were ascribed to HTLV-III, and not to HTLV-I. No phenotypic mixing between HTLV-III and HTLV-I was discerned, although MT-4 cells were HTLV-I-producer cell line. Thus, HTLV-III-infected MT-4 cells are thought to be useful in further study of the interaction between host cells and the virus, and appear to be a good viral source for the analysis of the virus.