The reliability and sensitivity of an indirect avidin-biotin-peroxidase (ABC) procedure for enumerating lymphocyte subpopulations was compared to flow cytometry (FC) employing direct immunofluorescence. Lymphocytes were enumerated by two different methods. For counting method I, which is the method of conventional FC, the number of immunostained lymphocytes was compared to the total number of lymphocytes present. The ABC procedure by method I detected a greater proportion of immunostained lymphocytes for all subsets tested than did FC. By counting method II, where the number of immunostained lymphocytes is compared to the total number of cells present, the ABC analysis still detected more total T cells than FC but the results for the two analyses were similar for T helper and T suppressor cells. Thus, the ABC technique appears to be a valid method for enumerating T lymphocyte subsets. Furthermore, as compared to FC, it offers the advantages of reduced cost, simplicity of understanding and performance, need for fewer cells, and a permanent record of lymphocyte staining. For these reasons, we feel that the ABC technique will enjoy widespread application for the identification of lymphocyte membrane antigens.