African green monkey (AGM) tripartite motif protein (TRIM) 5alpha can inhibit both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIVmac, whereas cynomolgus monkey (CM) TRIM5alpha can inhibit HIV-1, but not SIVmac. We previously reported that the 17-amino-acid region and an adjacent 20-amino-acid duplication in the SPRY(B30.2) domain of AGM TRIM5alpha determined the species specificity. In the present study, we demonstrated that CM TRIM5alpha had a dominant-negative effect on the anti-SIVmac activity of AGM TRIM5alpha. In contrast, mutant TRIM5alphas lacking the 20-amino-acid duplication did not have the dominant-negative effect, even though they failed to restrict SIVmac. These results indicated that oligomerization of the SPRY domain is required for anti-SIVmac activity and suggest that tight interaction between the viral capsid and all three molecules in one TRIM5alpha trimer may not be necessary for restriction activity.