Prolactin (PRL) is the primary lactogenic pituitary hormone that plays an essential role in many aspects of reproduction, from fertilization to mammary gland development and maternal behavior. PRL has also been reported to play a role in immunoregulation. Because initial observations indicated that hypophysectomized rats present abnormalities of the immune system, including increased thymic atrophy and lymphopenia, a number of studies have focused on the potential immunomodulatory roles of PRL. This hormone exerts its biological activities following binding to specific cell surface PRL receptors (PRLRs). In this report, we have characterized the development and function of the immune system in PRLR-deficient mice. Compared with wild-type control mice, PRLR−/− mice demonstrate no alterations in thymic or splenic cellularity or in the composition of the lymphocyte subsets present in primary (bone marrow and thymus) or secondary (spleen and lymph nodes) lymphoid organs. Lymphocytes from PRLR−/− mice are functional in vitro, as they can proliferate normally to mitogens, cytokines, and allogeneic cells. PRLR−/− splenocytes display normal NK-mediated cytotoxicity to YAC-1 target cells. In vivo studies have revealed that PRLR−/− mice are able to 1) generate normal steady-state Ig levels, 2) mount a normal specific Ig response following immunization with a T-dependent Ag, 3) eliminate injected allogeneic tumor cells, and 4) effectively control Listeria monocytogenes infection. Taken together, these results show that immune system development and function proceed normally in the absence of PRL-mediated signaling and suggest that PRLR pathways are not essential for immunomodulation in vivo.