We describe here the use of a sensitive and accurate multiprobe V beta RNase protection assay in characterizing the expression levels of 17 V beta genes in separated CD4+ and CD8+ subsets of selected mouse strains. The IE-reactive V beta genes (V beta s 11, 12, 5.1 and 16) showed various patterns of skewed subset expression in different strains, suggesting additional influences of IA, class I, and non-MHC genes in the selection process. Clonal deletion of V beta 11- and V beta 12-bearing T cells, among others, was skewed strongly towards the CD4+ subset in many IE+ mouse strains, supporting the notion that negative selection can cause incomplete, subset biased, V beta clonal deletions. Broad analysis in separated CD4+ and CD8+ subsets gave improved resolution of V beta repertoire selection, and revealed significant strain and/or subset specific skewing for additional V beta genes; with consistent bias towards higher expression of V beta 7 and V beta 13 in the CD8+ subset, and V beta 15 in the CD4+ subset of most mouse strains. The influence of diverse non-MHC ligands in V beta repertoire selection was further illustrated by the identification of unique V beta repertoires for six different MHC-identical (H2k) strains. Such polymorphisms in TCR repertoire expression may help to define better disease susceptibility phenotypes.