Cytotactin is an extracellular glycoprotein that influences neuron-glia interactions. It has been shown to appear in multiple forms that are differentially expressed in neural and non-neural tissues during vertebrate development. We report here the isolation and characterization of a cytotactin cDNA clone (lambda C801) that encodes 933 amino acids, equivalent to about half of a cytotactin polypeptide. Clone lambda C801 is an authentic cytotactin cDNA: it encodes a polypeptide that reacts with a monoclonal anti-cytotactin antibody and its deduced amino acid sequence is identical for 15 amino acids to the directly determined sequence of a CNBr fragment that reacted with the same antibody. Southern blot analyses with fragments of lambda C801 suggested that there may be only one cytotactin gene, but RNA transfer blots detected multiple mRNAs ranging in size from 6.5 to 8.0 kilobases. An 8.0-kilobase message and a Mr 240,000 cytotactin polypeptide were present in embryonic gizzard but not brain, while a 7.2-kilobase message and a Mr 220,000 polypeptide were present in brain but not gizzard. These results indicate that differential splicing of primary transcripts of the cytotactin gene yields various site-specific polypeptides. Sequence analyses of lambda C801 indicated that it specifies a region with extensive similarities to other proteins: the sequence begins with four consecutive epidermal growth factor-like repeats that are followed by eight segments that closely resemble each other and the type III repeats in fibronectin, and it ends with a 66 amino acid sequence similar to part of the beta and gamma chains of fibrinogen. One fibronectin-like repeat contains a single Arg-Gly-Asp sequence. The similarities with all three of these apparently unrelated proteins are extensive, suggesting that cytotactin has an evolutionary and possibly a functional relationship to each.