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The Ly‐6 alloantigens have been shown to play a critical role in T lymphocyte activation. To isolate a Ly‐6 cDNA, synthetic oligonucleotides, based on the partial amino acid sequence of purified Ly‐6E.1 protein, were used to probe a cDNA library. The synthetic oligonucleotides or the isolated cDNA detected a 1.1‐kb RNA species. Sequence analysis of the cDNA clone revealed that the Ly‐6E.1 protein consists of a 26‐amino acid leader followed by a 108‐residue, cysteine‐rich, core protein with no N‐linked glycosylation sites. Southern blot analysis of genomic DNAs revealed multiple bands indicating a family of related genes. Using recombinant inbred and Ly‐6 congenic strains of mice, restriction fragment length polymorphisms were demonstrable, and correlated with the Ly‐6 allotype of the DNA donors. This probe will enable further molecular genetic analysis of the role of Ly‐6‐linked proteins in the process of T lymphocyte activation. Isolation of Ly‐6 genomic clones may promote a further understanding of the complex tissue‐specific expression patterns characteristic of Ly‐6‐linked genes.