Human immunodeficiency virus type 2 (HIV-2) strains vary widely in their abilities to grow in Old World monkey (OWM) cells such as those of cynomolgus monkeys (CM). We evaluated eight HIV-2 isolates for their sensitivities to CM TRIM5alpha, an anti-HIV factor in OWM cells. We found that different HIV-2 isolates showed differences in their sensitivities to CM TRIM5alpha. Sequence analysis showed that TRIM5alpha-sensitive viruses had proline at the 120th position of the capsid protein (CA), whereas TRIM5alpha-resistant viruses had either alanine or glutamine. Mutagenesis studies indicated that the single amino acid at the 120th position indeed affected the sensitivity of the virus to CM TRIM5alpha.