Peptidoglycan hydrolases that are specifically associated with bacterial conjugation systems are postulated to facilitate the assembly of the transfer apparatus by creating a temporally and spatially controlled local opening in the peptidoglycan layer. To date little is known about the role of such enzymes in conjugation systems from Gram-positive bacteria. Conjugative plasmids from the Gram-positive pathogen Clostridium perfringens all encode two putative peptidoglycan hydrolases, TcpG and TcpI, within the conserved tcp transfer locus. Mutation and complementation analysis was used to demonstrate that a functional tcpG gene, but not the tcpI gene, was required for efficient conjugative transfer of pCW3. Furthermore, it was also shown that each of the two predicted catalytic domains of TcpG was functional in C. perfringens and that the predicted catalytic site residues, E-111, D-136, and C-238, present within these functional domains were required for optimal TcpG function. Escherichia coli cells producing TcpG demonstrated a distinctive autoagglutination phenotype and partially purified recombinant TcpG protein was shown to have peptidoglycan hydrolase-like activity on cognate peptidoglycan from C. perfringens. Based on these results it is suggested that TcpG is a functional peptidoglycan hydrolase that is required for efficient conjugative transfer of pCW3, presumably by facilitating the penetration of the pCW3 translocation complex through the cell wall.