During insulin-dependent diabetes mellitus, beta cell destruction may involve activation of the Fas-Fas ligand (Fas-FasL) system. Here, we employed dual-label immunohistochemistry to examine the intra-islet expression, distribution, and cellular sources of Fas and FasL in the NOD mouse. Pancreatic tissues were studied during spontaneous diabetes (days 21, 40, and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11, and 14 after cyclophosphamide administration). Our results show that FasL was expressed constitutively in most beta cells of NOD mice and in nondiabetes-prone mice, but not in glucagon or somatostatin cells or in islet inflammatory cells. It paralleled the loss of insulin immunolabeling with advancing disease. Immunolabeling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and nondiabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than in CD4 and CD8 cells. In the cyclophosphamide group, Fas expression in intra-islet CD4 and CD8 cells showed an increase close to the onset of diabetes. At days 11 and 14, several intra-islet macrophages with immunolabeling for Fas also coexpressed interleukin-1beta and inducible nitric oxide synthase. Fas was not detected in beta cells and other endocrine cells during spontaneous and cyclophosphamide diabetes. We show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. The role of Fas-FasL in beta cell destruction in the NOD mouse requires further clarification.