The rabbit cell line RL-5 was recently found to express only a single detectable major histocompatibility complex class I protein, in contrast to the multiple class I proteins expressed in cells of the mouse (K, D, and L) and human (A, B, and C). To clarify this difference in the rabbit major histocompatibility complex, we have begun an investigation of the class I genes expressed in the RL-5 cell line and here report the construction of 31 class I cDNA clones derived from RL-5 mRNA. Restriction endonuclease mapping has allowed classification of these clones into five groups, apparently representing five distinct class I mRNA transcripts. The sequence of a full-length cDNA from the predominant group (representing 26 of the 31 clones) reveals a typical class I structure, with an amino terminus that exactly matches the 36 amino terminal residues previously determined for the class I protein immunoprecipitated from RL-5. Complete sequence of a second distinct class I mRNA transcript was deduced from cDNA clones of the second largest group (representing two of the 31 clones). Although this transcript corresponds to a complete class I sequence, an anomaly in initiation sequence may preclude its translation in the proper reading frame. Comparison of the rabbit class I sequences to those of mouse and human revealed significantly higher homologies between the rabbit and the human than between mouse and rabbit or mouse and human sequences.