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Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in amino acid changes. Variation in SU was not random but occurred predominantly in five discrete regions. Within these variable regions, a remarkable 98% of the nucleotide substitutions changed the amino acid. These results demonstrate that extensive sequence variability accumulates in vivo after infection with molecularly cloned virus and that selection occurs in vivo for changes in distinct variable regions in env.