HIV-1 sequences from two possible transmission cases in South Africa were examined for evidence of genetic linkage. HIV-1-seropositive blood samples were obtained from a donor and recipient within 8 months following a blood transfusion and from a healthcare worker and her patient within 10 months following a needle-stick injury. A 700-bp region in env and 550-bp region in gag were analyzed. All sequences were phylogenetically associated with HIV-1 subtype C, the predominant HIV-1 subtype in South Africa. The nucleotide sequences from the blood transfusion case grouped together significantly with a bootstrap value of 100%. These samples were 98% and 100% identical in the predicted amino acid sequences of env and gag, respectively. In contrast, sequences from the needle-stick case showed only 67% and 80% amino acid identity in env and gag, respectively, and were separated on a phylogenetic tree. Molecular analysis suggested that HIV transmission occurred in the blood transfusion case but not in the case of the needle-stick injury. These data emphasize the need for molecular investigation of epidemiologically linked cases of HIV transmission.