Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxytransferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies. p iaOGrtAMMED cell death 0~ D) 1 is a selective process of physiological cell deletion (Wyllie, 1981; Umansky, 1982; Bursch et al., 1990; Ucker, 1991). Its execution plays a major role in the control of shape and size in normal and abnormal processes (Kerr et al., 1972, 1987; Martz and Howell, 1989; Williams, 1991; Ucker, 1991). PCD exerts a homeostatic function in relation to tissue dynamics, as the steady state of continuously renewing tissues is achieved by a balance between cell replication and cell death. While cells active in DNA synthesis can be specifically labeled in situ by [3H] thymidine, mlUdR, or BrdU incorporation, the lack of an appropriate methodology for the identification of PCD in situ hampers the research of tissue kinetics (Flume, 1987; Allen, 1987; Hirshfield, 1988; Rothenberg, 1990; Tadakuma et al., 1990; Motyka and Reynolds, 1991). Most of our present knowledge about the nature of programmed cell death has come from studies with cells in culture, mainly lymphocytes, in systems where PCD was experimentally induced (reviewed by Allen, 1987; Martz and Howell, 1989; Williams, 1991; McConkey et al., 1990; Ucker 1991). These experiments showed that PCD is associated with endogenous endonuclease activity (Wyllie, 1980; Wyllie et al., 1984; Cohen and Duke, 1983, 1984; McConkey et al., 1989, 1990; Shi et al., 1990; Brune et al., 1991). It seems that chromatin cleavage is the most characteristic biochemical feature of the process (Wyllie et al., 1984; Kerr et al., 1987; Martz and