Natural killer (NK) cells are potent cytotoxic and cytokine producing innate lymphocytes that recognise and respond to infected or transformed cells. Their function and specificity are controlled by activating and inhibitory receptors, often including expanded and diversified receptor families such as KIR (killer cell immunoglobulin-like receptors) and KLR (killer cell lectin-like receptors). These receptor families have convergently and divergently evolved during mammalian radiation, with the most common ligands being MHC-I. However, ligands for these expanded receptor families remain enigmatic outside of primates. Cattle are unique amongst species currently studied as they have expanded both KIR and KLR, although this is likely to be the case in other ruminant species. As previous analysis had indicated the likely co-evolution of bovine KIR and bovine MHC-I (BoLA-I), we attempted to discover and characterise KIR and BoLA-I interactions and further investigate cattle NK cell heterogeneity to complement this investigation. Bovine KIR (bota-KIR) were expressed as tetramers (bota-KIR-tet: 2DL1) or Fc fusion proteins (bota-KIR-Fc: 2DL1 or 3DXL1). For botaKIR2DL1, correct protein conformation was validated with a bota-KIR2DL1 specific antibody. No equivalent reagent was available for bota-KIR3DXL1. KIR-tete and KIR-Fc did not bind to a panel of P815 cell lines expressing nine phylogenetically diverse BoLA-I alleles in flow cytometry. In a series of experiments with bovine PBMC, KIR-Fc and KIR-tet bound to B-cells and NK-cells respectively. Attempts to identify the exact protein mediating these interactions, through a combination of western blotting, immunoprecipitation and BLI analysis, has proved unsuccessful. 10X genomics analysis to investigate cattle NK cell heterogeneity has indicated a highly diverse population with multiple distinct sub-clusters. KIR and KLR expression were detected but at too low a level to relate expression back to likely NK function. Together these data indicate that the bovine NK cell population is highly heterogeneous, and suggests that the ligand for bovine KIR is not BoLA-I. It may also indicate that the expanded KLR receptors act as the primary BoLA-I receptor in cattle, as is the case in lemurs and mice. Elucidating ligand interactions for the KIR and KLR in bovine continues to be important for our fundamental understanding of bovine immune responses.