Because of positive and negative selection to molecules of the major histocompatibility complex (MHC), only a small proportion of the massive numbers of T cells generated in the thymus are selected for export. Immature thymocytes have a rapid turnover, and it has long been assumed that most thymocytes die in situ, presumably from apoptosis. This has yet to be proved, however, and conventional staining techniques have shown only minimal evidence of cell death in the normal thymus. Using a method for detecting cells with DNA strand breaks, we now present direct evidence for apoptosis in the normal thymus. In sections of thymus from adult mice, apoptotic cells are scattered throughout the cortex and are engulfed locally by F4/80+ macrophages. Apoptosis in the thymic cortex is not reduced in MHC-deficient mice, which suggests that T-cell death is primarily a reflection of lack of positive selection rather than negative selection. Direct evidence for apoptosis due to negative selection was obtained by crossing a V beta 5 transgenic line to I-E+ and I-E- mice: I-E+ mice are known to eliminate V beta 5+ T cells in the thymus whereas I-E- mice do not. In marked contrast to I-E- mice, the medulla of I-E+ V beta 5 transgenic mice contains dense aggregates of apoptotic cells; these cells are engulfed by a distinct population of F4/80- MAC-3+ macrophages. Negative selection of V beta 5+ cells is thus restricted to the medulla.