We have analyzed tolerance-related clonal deletion of Mls-and I-E-reactive thymocytes at the RNA level using a multi-V beta probe RNAse protection assay, and used this phenomenon to identify the maturation stage of the abnormally expanded CD4-8-, TCR-alpha/beta + subset in lpr and gld homozygous mice, and of the phenotypically similar minor thymocyte subset found in normal mice. Essentially complete V beta clonal deletions were detected in lpr and gld cells of all appropriate background strains. Substantial, but not complete, V beta clonal deletions were also detected in the CD4-8- TCR-alpha/beta + subset of normal mice. Since expression of CD4/CD8 is required for V beta clonal deletions to occur, we conclude that lpr and gld cells, and at least a portion of CD4-8- TCR-alpha/beta + thymocytes in normal mice, are derived by secondary loss of CD4/CD8 accessory molecules from more mature CD4+8+ precursors. One possible interpretation of these findings is that such CD4/CD8 loss may affect a class of self-reactive thymocytes that have escaped direct clonal deletion. Exportation and expansion of such cells in the periphery may be an important contributory factor in the induction of systemic autoimmunity.