TRIM5␣ mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5␣. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.One of the major determinants of host restriction of retroviral replication is the longest (alpha) isoform of the host protein TRIM5, a member of the tripartite motif protein family (34, 42). Human TRIM5␣ (huTRIM5␣) inhibits preintegration stages of murine leukemia virus N-strain (MLV-N) infection, while rhesus macaque TRIM5␣ inhibits human immunodeficiency virus type 1 (HIV-1) infection (13,16,55). Recent studies have demonstrated retroviral restriction by TRIM5␣ species-specific variants from cows (Bos taurus) (41, 56) and rabbits (Oryctolagus cuniculus) (38), suggesting a common ancestor for mammalian TRIM5␣ with antiretroviral properties. Tripartite motif proteins typically comprise a RING domain with E3-ubiquitin ligase activity capable of autoubiquitination (54), a B-box 2 domain, and a coiled-coil domain, collectively know as the RBCC (34). Additionally, some TRIM proteins, including TRIM5␣, possess a C-terminal B30.2 (PRY-SPRY) domain. TRIM5␣ blocks reverse transcription (RT) in most nonpermissive cells (16,42), and evidence suggests that TRIM5␣ homodimers engage the incoming retroviral capsid in the cytoplasm via the B30.2 domain (18,24,40,44). The resulting complex is then degraded rapidly by the proteasome (9, 47) (proteasomal inhibitors restore RT but not viral replication [3,53]). Human splice variants TRIM5␦ and TRIM5␥ lack a B30.2 domain, which disrupts their ability to restrict (30,42). Moreover, these short TRIM5 isoforms have a dominant negative effect, impairing the activity of fulllength TRIM5␣, presumably by the formation of heterodimers (24, 32).