Retroviral restriction factors are cellular proteins that interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle [1–3]. The first restriction activity described was the mouse Fv1 gene [4]. Three different alleles of Fv1, capable of restricting different murine leukaemia viruses (MLV), have been characterized at the molecular level [5, 6]. Two further activities, Ref1, which acts on MLV, and Lv1, which acts on lentiviruses, have been identified in other mammalian species [7–9]. Recently, it has become clear that Ref1 and Lv1 are encoded by the same gene, Trim5α, which inhibits retrovirus replication in a species-specific manner [10–14]. A series of chimeras between the human and rhesus monkey Trim5 genes were created to map and identify these specificity determinants. The Trim5α SPRY domain was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY domain. A single amino acid substitution (R332P) in the human Trim5α can confer the ability to restrict HIV-1, suggesting that small changes during evolution may have profound effects on our susceptibility to cross-species infection.