Cryptdins constitute a diverse population of defensins in Paneth cells of intestinal crypts. In mice, certain intestinal mRNAs, termed "CRSIC" and "CRS4C," are considered to be cryptdin-related sequences, because their prepro-coding sequences are 94% identical to those of cryptdin-1 mRNA; however, their predicted products, which are cationic, cysteine-rich peptides are not defensins (A. J. Ouellette and J. C. Lualdi, J. Biol. Chem. 265: 9831-9837, 1990). Here we describe several mouse small intestinal mRNAs and genes that code for CRS4C prepropeptides. The 10-kDa deduced CRS4C proteins consist of a prepro sequence, potential monobasic or dibasic peptide cleavage sites, a predicted 3.7-kDa peptide that contains 7 [C]-[X]-[Y] repeats, and a C(N/K)CNPK carboxyl-terminal consensus sequence. In situ hybridization experiments showed that CRS4C mRNAs are found in Paneth cells of adult small bowel. The CRS4C genes closely resemble cryptdin genes, having a two-exon structure with highly conserved transcription start sites, intron-exon junctions, and a single intron of approximately 550 bp. Like the cryptdin genes, exon 1 of CRS4C genes consists of 5' untranslated sequences (UTS) and the prepro-coding region, and exon 2 codes for the predicted mature peptide and 3' UTS. Despite the similarity of first exons in CRS4C and cryptdin genes, their introns exhibit very little homology, and their second exons code for unrelated peptides. Analysis of introns suggests that the ancestral cryptdin and CRS4C genes may have diverged from a common ancestor in the distant past and expanded only recently. We speculate that the cryptdin/CRS genes evolved so that prepro regions encoded by exon 1 were conserved to allow the varied peptides coded by exon 2 to be directed into Paneth cell secretory granules.