The retroviral restriction factor, TRIM5alpha, blocks infection of a spectrum of retroviruses soon after virus entry into the cell. TRIM5alpha consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The B-box 2 domain is essential for retrovirus restriction by TRIM5alpha, but its specific function is unknown. We show here that the B-box 2 domain mediates higher-order self-association of TRIM5alpha(rh) oligomers. This self-association increases the efficiency of TRIM5alpha binding to the retroviral capsid, thus potentiating restriction of retroviral infection. The contribution of the B-box 2 domain to cooperative TRIM5alpha association with the retroviral capsid explains the conditional nature of the restriction phenotype exhibited by some B-box 2 TRIM5alpha mutants; the potentiation of capsid binding that results from B-box 2-mediated self-association is essential for restriction when B30.2(SPRY) domain-mediated interactions with the retroviral capsid are weak. Thus, B-box 2-dependent higher-order self-association and B30.2(SPRY)-dependent capsid binding represent complementary mechanisms whereby sufficiently dense arrays of capsid-bound TRIM5alpha proteins can be achieved.