B lymphocytes bearing the Leu-1 cell-surface antigen (Leu-1⁺), the human equivalent of mouse Ly-1⁺ B lymphocytes, have been detected in human peripheral blood, but there is little information on their frequency and properties. Analysis by fluorescence-activated cell sorter and double immunofluorescence showed that Leu-1⁺ B cells are consistently present in the peripheral blood and spleens of healthy subjects and constitute 17.0 ± 5.0% (mean value ± standard deviation) and 17.3 ± 3.9%, respectively, of total B cells. When purified Leu-1⁺ and Leu-1^- B lymphocytes were transformed into immunoglobulin-secreting cells by infection with Epstein-Barr virus and the culture fluids were tested for reactivity with self-antigens, at least two important autoantibodies, antibody to the Fc fragment of human immunoglobulin G (rheumatoid factor) and antibody to single-stranded DNA, were found to be made exclusively by Leu-1⁺ B cells. It is concluded that the Leu-1⁺ lymphocytes represent a major subset of the normal human B cell repertoire and include the B cells capable of making autoantibodies similar to those found in systemic lupus erythematosus and rheumatoid arthritis.