cDNA clones coding for a T-cell receptor beta-chain were isolated from a beef insulin/IAd-reactive T-cell hybridoma, A20.2.15, and its complete amino acid sequence was deduced. This beta-chain gene utilizes the same V beta segment as a thymocyte beta-chain gene (86T1) and rearranges to the 5' proximal J-C locus (J beta 1-C beta 1), thus providing definitive evidence of a beta-chain gene from a functional hybridoma that utilizes C beta 1. The amino acid sequence of the V beta gene in A20.2.15 is identical to 86T1, thus suggesting the absence of somatic mutation in the beta-chain of A20.2.15. Southern blot analysis revealed a somatic DNA rearrangement unique to the A20.2.15 hybridoma. The expression of this gene in the hybridoma was confirmed by RNA dot hybridization. All 24 beta-chain clones so far isolated from the A20.2.15 hybridoma contained C beta 1, suggesting that the beta-chain gene of the fusion partner BW5147 is not expressed in the hybridoma.