Although a small amount of viral DNA has been shown to be enclosed within human immunodeficiency virus type 1 (HIV-1) virions, the majority of full-length viral DNA is formed after this virus infects target cells. Hence, we undertook investigations to identify the physical characteristics of the HIV-1 replication unit during the early events of infection. In these studies, nascent viral DNA synthesis was found to occur between 15 and 30 min after purified, DNase-treated HIV-1 virions were added to HUT 78 cells. At 1 h postinfection, a large amount of strong-stop viral DNA and some first-strand viral DNA had been synthesized. Several lines of evidence, including purification, nuclease digestion, and immunoprecipitation, indicated that these nascent viral DNAs were located within particles containing components such as reverse transcriptase and p24gag and gp120env proteins and having physical characteristics similar to those of intact virions.