Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1⁺ CD3⁻ cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1⁺ CD3⁻ cells were responsible for acute tumor rejection, establishing the relationship of NK1.1⁺ CD3⁻ cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNγ in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.