T cell immunoglobulin and mucin (Tim) family molecules are cell membrane proteins with four functional Tim family members in mouse, and three in human. They are preferentially expressed on immune cells with Tim1 on Th2 cells, Tim3 on Th1 cells and Tim4 on antigen-presenting cells (APCs). They have several roles, including regulating immune responses and mediating phagocytosis of dead cells. However, little is known about them beyond these two species, and nothing outside mammals. To investigate the Tim family in the chicken, the genes were identified and cDNAs cloned. Differently to mammals, the chicken genome only contains genes for Tim1 and Tim4. Chicken Tim1 (chTim1) has similar mRNA expression patterns to those of mammalian Tim1 in lymphoid tissues and immune cells. Interestingly, chTim4 has at least four splice variants – an extra short isoform (chTimeS) lacking exons 5, 6, 7 and 8, a short isoform (chTim4S) without exons 3, 4 and 5, a long isoform (chTim4L) with all exons and an extra long isoform (chTim4eL), which is similar to chTim4L but with a longer exon 3. The chTim4S is a homologue of mammalian Tim4 with constitutive expression in lymphoid tissues and immune cells; other chTim4 variants showed inducible or cell-specific expression patterns. Like mammalian Tim4, chTim4S is expressed by APCs; but differently to mammals, chTim4S is also expressed by γδ T cells, suggesting a unique role for chTim4 in this population of T cells. The biological activities of the chicken Tim family molecules were also investigated using chTim-Ig fusion proteins. Like mammals, chTim1 and chTim4S fusion proteins can specifically recognise phosphatidylserine (PS), an indicator of apoptotic cells, suggesting they are PS receptors. Pre-incubation with PS blocked binding of the chTim4S fusion protein to PS-exposing apoptotic cells. Physiologically, recognition of PS by the chTim proteins mediates apoptotic cell clearance, which was demonstrated using chTim-transfected fibroblast cells (3T3), which significantly increased their uptake of apoptotic cells compared with untransfected cells. The chTim4-Ig fusion protein also had costimulatory activity on chicken T cells. Monoclonal antibodies against the chTim proteins were generated. They specifically recognise their own antigen tested intensively by different immunological assays. ChTim4L is expressed intracellularly in freshly-isolated splenocytes rather than on the surface, whereas PMA-stimulated splenocytes express chTim4S and chTim4L on the cell surface. Like mammals, chicken splenic macrophages also express chTim4S and chTim4L. Both of them are also expressed by bone marrow-derived macrophages but not bone marrow-derived DCs. The chTim1 protein was detected at high levels in bursal cells and splenocytes by western blot analysis using polyclonal anti-chTim1 serum, which is consistent with its mRNA expression pattern through qRT-PCR analysis, suggesting B and T cells may express chTim1, consistent with its expression in mammals. Mammalian Tim1 is expressed on Th2 cells, its ligand, Tim4, on APCs; the interaction between them drives Th2 cell proliferation. The knowledge from this study will help to further dissect how the chicken’s Th2 responses are regulated through cell surface molecules.