Many bacteria express a surface-exposed proteinaceous layer, termed the S-layer, which forms a regular two-dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S-layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S-layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post-translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high-MW SLP shows weak homology to N-acetyl muramoyl-L-alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high-MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high-MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis to be transcribed.