Cytofluorometric analysis using specific monoclonal antibodies directed against the T cell antigens Thy-1.2, CD4, CD8, CD4V/J(8.1+8.2+8.3), and the antigen Mac-1 expressed by mature macrophages and NK cells were used to characterize and quantify the phenotypes of (1) unfractionated and Percoll gradient fractionatedin situislet immune cells isolated from prediabetic and diabetic female NOD mouse spleens. We found in prediabetic female mice that the majority (-70%) of thein situislet immune cells were Thy-1.2 positive T cells. CD4 positive T cells (-40%) were the most abundant phenotype together with double negative T cells (-20%). The percentages of CD8 positive T cells were -10%, and only -4% of the immune cells were Mac-1 positive. The percentages of CD4V/3(8.1+8.2+8.3) positive and double negative T cells in diabetic spleens were significantly higher in comparison to prediabetic spleens. In C57B1/6J control nondiabetic mice the percentage of double negative T cells in the spleens was significantly 4-fold lower when compared to diabetic NOD spleens. The specific cytolytic activity mediated byin situislet immune cells against 51Cr-labeled dispersed syngeneic single-cell islet cells at an effector to target ratio of 20 was twenty- to thirty-fold higher than that mediated by prediabetic splenic lymphoid cells. It is concluded that prediabetic NOD mousein situislet immune cells are mostly CD4 positive and double negative T cells, and that CD4 and CD8 positive T cells in the intra-islet infiltrate warrants further evaluation as potential effector T cells in target J-cell destruction.