A 14.6 kb prophage-like insertion, termed skinCd, was found to interrupt the sigK gene, which encodes an RNA polymerase sigma factor essential for sporulation, in six strains of Clostridium difficile. Until now, Bacillus subtilis was the only spore-former shown to carry such an insertion, and the presence of the insertion is not required for efficient sporulation in this organism. The B. subtilis and C. difficile skin elements proved to be divergent in sequence, inserted at different sites within the sigK gene and in opposite orientations. The skinCd element was excised from the chromosome specifically during sporulation, forming a circular molecule. Two natural isolates of C. difficile lacked the skinCd element and were defective in sporulation. When a merodiploid strain was created that carries both interrupted and uninterrupted versions of the sigK gene, the cells became Spo-, showing that the uninterrupted gene is dominant and inhibits sporulation. C. difficile sigK genes, whether skinCd+ or skinCd-, lack the N-terminal pro-sequence found in all other sigK genes studied to date. Thus, regulated excision of skinCd appears to be a critical mechanism for achieving proper temporal activation of sigmaK.