Two cynomolgus macaques were infected with a complex, but characterized, challenge stock of simian immunodeficiency virus (SIVmac251 32H). The polymerase chain reaction was applied in a temporal sequence analysis to determine the sequences of the gp120 region of the SIV env gene, which were present in the blood of both macaques at 1, 6, and 15 months postinfection (p.i.). At 1 month p.i. selected sequences, which had been present in the original virus challenge stock, were reisolated. At later times, new sequences emerged, which had not been detected in the original virus challenge stock. Changes in sequence were restricted to specific regions of gp120, notably those equivalent to V1, V2, V4, and V5 of HIV-1, but not V3. The diversity and the rate of appearance of new sequences in the V1 region suggest that genetic evolution occurs by mechanisms in addition to nucleotide substitutions. These results are discussed in relation to the role of the envelope protein in the generation of protective immunity against infection with immunodeficiency viruses.