ABSTRACT TRIM5 is a determinant of species-specific differences in susceptibility to infection by retroviruses bearing particular capsids. Human immunodeficiency virus type 1 (HIV-1) infection is blocked by the alpha isoform of macaque TRIM5 (TRIM5α rh ) or by the product of the owl monkey TRIM5-cyclophilin A gene fusion (TRIMCyp). Human TRIM5α potently restricts specific strains of murine leukemia virus (N-MLV) but has only a modest effect on HIV-1. The amino termini of TRIM5 orthologues are highly conserved and possess a coiled-coil domain that promotes homomultimerization. Here we show that heterologous expression of TRIM5α rh or TRIMCyp in human cells interferes with the anti-N-MLV activity of endogenous human TRIM5α (TRIM5α hu ). Deletion of the cyclophilin domain from TRIMCyp has no effect on heteromultimerization or colocalization with TRIM5α hu but prevents interference with anti-N-MLV activity. These data demonstrate that TRIM5 orthologues form heteromultimers and indicate that C-terminal extensions alter virus recognition by multimers of these proteins.