Studies presented here demonstrate the expression of the Ly-l gene and the detection of the Ly-l cytodifferentiation antigen in murine hybridomas producing autoantibodies. We examined the transcription of the Ly-I gene in thymocytes and 140 hybridomas producing autoantibodies of various specificities which were obtained from normal and autoimmune disease prone mouse strains. As previously demonstrated thymocytes stain brightly for Ly-l by immunofluorescence and express Ly-l transcripts. In our panel of hybridomas producing autoantibodies Ly-l transcripts were detected in 31 (45%) out of 69 NZB hybridomas and 7 (88%) out of 8 viable motheaten hybridomas. S1 nuclease protection experiments showed that Ly-l transcripts detected in thymocytes and B cells are the product of the same gene. The B cell transcripts are functional since immunofluorescence and Western data presented here detected the Ly-I protein in hybridomas cells which were found to transcribe the Ly-l gene. Interestingly a polymorphic transcription of the Ly-l gene was observed in B cells and B cell hybridomas as compared to thymocytes. Our results obtained in the hybridoma system firmly establish a major contribution of the Ly-l B cell subset to the production of DNA specific autoantibodies and a smaller contribution to the production of rheumatoid factors and“natural”, multispecific autoantibodies.