The F1–20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1–20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1–20 protein. We found two distinct isoforms of F1–20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1–20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3′ acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1–20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1–20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1–20 protein. We then determined biochemically that the F1–20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1–20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1–20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1–20 protein is phosphorylated in vivo.