A cDNA containing the complete open-reading frame encoding rabbit antithrombin III (AT-III) was isolated from a rabbit liver cDNA expression library, using a specific antibody as a probe. Sequence analysis showed 84% identity between the deduced amino acid sequences of the rabbit and human proteins. A previously described cell-free expression system was used to verify the identity of the clone. The full-length cDNA was inserted into an expression vector, and messenger RNA (mRNA) transcripts generated. In vitro translation of these transcripts, in the presence of [35S]methionine, in an mRNA-dependent rabbit reticulocyte lysate system resulted in the synthesis of a 51-Kd polypeptide, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This nonglycosylated protein was capable of forming SDS-stable complexes with human alpha-thrombin. Complex formation was significantly enhanced following the deletion of nucleotides encoding the signal peptide, and the resultant generation of a 47-Kd nonglycosylated mature protein product. When the template DNA giving rise to this product was internally truncated, two rabbit AT- III deletion mutants were generated that lacked the ability to interact with thrombin, but retained the ability to bind heparin. Cell-free expression plasmids encoding the human and rabbit AT-III mature molecules were manipulated to produce two interspecies fusion proteins. For the first, human codons were used to replace rabbit codons from residue 369–433, while in the second human codons replaced rabbit codons from residue 217–433. Both fusion proteins exhibited less efficient thrombin-complexing ability than the original cell-free- derived mature rabbit AT-III. Thus, portions of AT-III molecules from the two species, despite their high degree of homology, are not interchangeable. Knowledge of the structure of rabbit AT-III, combined with the availability of the rabbit cDNA, will permit defined experimentation aimed at understanding antithrombin III structure relative to its function in vivo.