A cDNA library of TCR beta chain transcripts from BALB/c thymocytes was constructed using anchored polymerase chain reaction (PCR). Screening of this library led to the identification of a V beta gene segment, V beta 20, structurally related to V beta 3 and V beta 17. Genomic analysis of mice displaying deletions in their V beta loci, together with mapping of cosmid clones, situated V beta 20 2.5 kb beside V beta 17. The expression of V beta 20 was estimated by PCR in mice of different H-2 and Mls types. Peripheral T cells from H-2k and H-2d mice did not express V beta 20, whereas in I-E-negative mice (C57Bl/6 and SJL), V beta 20 transcripts were detected. The lack of V beta 20 transcripts in (C57Bl/6 x CBA/J)F1, (C57Bl/6 x BALB/c)F1, and in congenic B6.H-2k mice suggests that the differential use of V beta 20 is due to an I-E-mediated clonal deletion process. The involvement of the Mls super antigens was excluded by analysis of all Mls type combinations. The nature of the V beta 20-deleting element(s) is discussed in the context of the I-E/superantigen systems controlling the expression of V beta 11 and V beta 17.