The tetracycline resistance determinant from pCW3, a conjugative plasmid from Clostridium perfringens, has been identified and the structural gene localized to within a 1.4-kb region. Hybridization analysis, which utilized an internal 0.8-kb specific gene probe, showed that eight nonconjugative tetracycline resistant C. perfringens strains all carried homologous resistance determinants. No homology was detected in DNA prepared from tetracycline resistant isolates of Clostridium difficile or Clostridium sporogenes. However, the one strain of Clostridium paraputrificum that was tested did contain an homologous determinant. No homology was found to any of the recognized classes of tetracycline resistance determinants. The C. perfringens tetracycline resistance determinant represents a new hybridization group, Class P.