Murine 2B4 (CD244) is a cell surface receptor expressed on all NK cells, γδ-T cells, a subset of CD8+ T cells, and all CD14+ monocytes. 2B4 binds to CD48 with high affinity, and cross-linking 2B4 with anti-2B4 Ab in vitro causes activation of NK cells. To study its physiological role, we have generated, by gene targeting, mice deficient in the expression of this cell surface molecule. The expression of lymphoid cell surface markers on PBMC and splenocytes of mice homozygous for the mutation in 2B4 (2B4−/−) is identical to that in wild-type mice. However, thymocytes from female 2B4−/− mice, but not male 2B4−/− mice, have an increase in the immature CD4−/CD8− population. To investigate the in vivo role of 2B4, wild-type and 2B4−/− mice were injected with CD48+ and CD48− metastatic B16 melanoma cells. Wild-type mice rejected CD48+ melanoma poorly compared with CD48− tumor cells, suggesting that ligation of 2B4 by CD48 on melanoma cells is inhibitory. In keeping with this, male 2B4−/− mice showed enhanced ability to reject CD48+ melanoma cells. However, female 2B4−/− mice poorly rejected both CD48+ and CD48− melanoma cells, revealing a gender-specific and CD48-independent defect in mice lacking 2B4. In vitro and in vivo experiments reveal a complex role of NK cells in gender specificity.