An extensive comparison of TCR alpha beta V-region usage by CD8 beta-CD4+CD8+ intraepithelial lymphocytes (IEL), CD4-CD8+ IEL, and lymph node (LN) T cell subsets in three minor lymphocyte stimulating (Mls)-disparate, MHC-identical mouse strains revealed novel TCR selection patterns. In cases where forbidden V regions were expressed by CD8 beta- CD4-CD8+ IEL, the same TCRs were deleted from CD8 beta- CD4+CD8+ IEL, indicating that lack of CD8 beta expression was not solely responsible for forbidden V-region expression. These results also suggested that CD4 may be involved in negative selection of CD4+CD8+ IEL TCRs. In C57BR/cdJ (Mls-1b2b) mice, a major increase in V beta 3+CD4+CD8+ IEL but not in other IEL or LN subsets was noted suggesting a subset-specific expansion of V beta 3+ cells. Negative selection of V beta 14+ cells in only the CD4+CD8+ IEL subset further supported the existence of intestine-specific TCR selection processes. Analysis of V-region expression of CD8 beta + and CD8 beta-CD4-CD8+ IEL subsets revealed that forbidden V-region expression was not strictly confined to the CD8 beta- subset in all cases. Overall, the data point to a dynamic, gut-specific TCR selection process that may be antigen driven.