Evidence for human immunodeficiency virus type 1 (HIV-1) superinfection was investigated among a group of four previously HIV-1 infected transfusion recipients (and the four implicated HIV-1 infected donors) identified by the Transfusion Safety Study, and two groups of 4 and 5 Brazilian injection drug users, who consistently injected themselves using shared paraphernalia. To probe these cases for possible superinfection we used heteroduplex mobility analysis (HMA) of HIV-1 tat, a technique which is a reliable for establishing epidemiologic linkages and searching for minor strains in mixed infection settings. In all these cases with established, untreated HIV-1 infections, we were unable to detect HIV-1 superinfection, even though the involved individuals were at high risk for second strain acquisition. We therefore conclude that although superinfection can occur in a few cases, it is a rare event, and the vast majority of recombinant HIV-1s characterized to date resulted from acute coinfections, rather than superinfection.