The phosphatidylinositol-linked T-cell surface protein RT6 occurs in two alloantigenic forms, RT6.1 and RT6.2. A lambda gt11 cDNA expression library was constructed from the RT6.2+ T-T hybridoma cell line EpD3 and screened with RT6-specific antisera. One of nine independent positive clones obtained in an initial screening of 500,000 recombinant phages was able to select antibodies capable of immunoprecipitating RT6.2 as well as RT6.1. Northern and Southern blot analyses with the cDNA insert of this clone provide additional strong evidence for its RT6.2 specificity. The 330 bp cDNA insert of this clone hybridizes with a 1.6 kb mRNA species present in RT6.2+ and RT6.1+ but absent from RT6- T-cell lines. In Southern blot analyses this insert detects a DNA restriction fragment length polymorphism between genomic DNAs from RT6.1 and RT6.2 rat strains. In addition, these analyses indicate that RT6.1 and RT6.2 are each encoded by a single gene and provide no evidence for the existence of additional RT6 alleles or a closely related gene family. Preliminary sequence analyses suggest that the RT6.2-specific partial cDNA found in these studies represents an internal EcoRI fragment of the corresponding cDNA and encodes part of the presumptive leader and the N-terminal domain of RT6.2. The immunological reactivity of the corresponding fusion protein indicates that the N-terminal domain of RT6.2 contains an allotype-specific epitope as well as at least one additional epitope shared with the alloantigenic counterpart RT6.1.