High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.