Natural killer (NK) cells recognize and kill certain tumor cells, virally infected cells and MHC class I-disparate normal hematopoietic cells. NK cell cytotoxicity is regulated by a multitude of receptors with either activating or inhibitory signaling function. We here report the molecular cloning of bovine CD94 [killer cell lectin-like receptor (KLR)-D1] and NKp46 orthologues, four members of a bovine CD158 [killer cell immunoglobulin-like receptor (KIR)] family, and a novel KLR. This novel receptor was termed KLRJ1 and is most similar to Ly-49 (KLRA). The KLRD1 and KLRJ1 loci were mapped to a bovine NK gene complex on chromosome 5 by radiation hybrid mapping, whereas KIR2DL1 and NKP46 were localized to chromosome 18. Two of the bovine KIR(KIR2DL1 and KIR3DL1) contain immunoreceptor tyrosine-based inhibition motifs (ITIM), suggesting an inhibitory function. Bovine KIR2DS1 and KIR3DS1 lack ITIM but have an arginine-containing motif in their transmembrane domain, similar to primate KIR2DL4. Thus, KIR multigene families with divergent signaling motifs do not only exist in primates. Based on sequence comparison, it appears that the primate and bovine KIR multigene families may have evolved independently.