The negative selection of T cells expressing the gamma delta T cell antigen receptor (gamma delta T cells) was studied using transgenic mice expressing a gamma delta receptor with specificity for an H-2T-linked class I major histocompatibility complex molecule from H-2b mice. The potentially self-reactive gamma delta thymocytes in H-2b/d transgenic mice are larger and have lower levels of gamma delta T cell receptor expression than gamma delta thymocytes from H-2d mice. H-2b/d gamma delta thymocytes do not respond to H-2b antigen-presenting cells, and thus are inactive compared to H-2d gamma delta thymocytes. However, the H-2b/d gamma delta thymocyte population, but not the H-2d gamma delta thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self-reactive gamma delta thymocytes undergo programmed cell death. This in vitro programmed cell death of self-reactive gamma delta thymocytes may reflect the clonal deletion process that results in a depletion of gamma delta T cells in the peripheral lymphoid organs of adult H-2b/d mice. We also present evidence that self-reactive gamma delta T cells, similarly to alpha beta T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.