Strains of the periodontal pathogen Campylobacter rectus express a 150- to 166-kDa protein on their cell surface. This protein forms a paracrystalline lattice, called the surface layer (S-layer), on the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the function of the S-layer in the pathogenesis of C. rectus, we have cloned and characterized its gene. The S-layer gene (crs) from C. rectus 314 encodes a cell surface protein which does not have a cleaved signal peptide at its amino terminus. Although the amino acid sequence deduced from the crs gene has 50% identity with the amino-terminal 30 amino acids of the four S-layer proteins from Campylobacter fetus, the similarity decreases to less than 16% over the rest of the protein. Thus, the crs gene from C. rectus encodes a novel S-layer protein whose precise role in pathogenesis may differ from that of S-layer proteins from other organisms. Southern and Northern blot analyses with probes from different segments of the crs gene indicate that the S-layer gene is a single-copy, monocistronic gene in C. rectus. RNA end mapping and sequence analyses were used to define the crs promoter; there is an exact match to the Escherichia coli 10 promoter consensus sequence but only a weak match to the 35 consensus element. Southern blots of DNA from another strain of C. rectus, ATCC 33238, demonstrated that the crs gene is also present in that strain but that there are numerous restriction fragment length polymorphisms in the second half of the gene. This finding suggests that the carboxy halves of the S-layer proteins from strains 314 and 33238 differ. It remains to be determined whether the diversities in sequence are reflected in functional or antigenic differences important for the pathogenesis of different C. rectus isolates.