The promoter sequences of individual murine TCR Vbeta segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCRbeta 3' enhancer. However, thymocytes of recombination-activating gene-2 (Rag2)-deficient mice allow the transcriptional activity of Vbeta promoters to be studied before rearrangement. Here we report that many Vbeta segments are detectably transcribed in Rag2(-/-) thymocytes and that there are significant differences in expression among different Vbeta segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline Vbeta transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal Vbeta cluster (Vbeta2.1, Vbeta1.1, Vbeta4.1) are relatively strongly expressed and more proximal Vbeta segments (Vbeta14.1, Vbeta3.1, Vbeta7.1, Vbeta6.1) are only weakly expressed. Different Vbeta segments also show different developmental programs of activation in different thymocyte subsets, with the Vbeta5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag(+) MHC class I(-/-) and class II(-/-) thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by beta selection, before MHC-dependent selection. However, the expression pattern of Vbeta2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.