Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11. These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs. DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N-CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs. A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N-CAM polypeptides. In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides. The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene.