The toxigenic element of Clostridium difficile strain VPI 10463 is identified by establishing boundaries between toxigenic sequences and those sequences shared by nontoxigenic and toxigenic strains. The toxigenic element is chromosomal, 19.6 kb in length, and comprised of five open reading frames which include the toxin A and B genes. Four of the open reading frames are contiguous and are transcribed in the same direction. The fifth is downstream from the others and oriented in the opposite direction. One of the open reading frames, located 5' to the toxin B gene, is previously unknown. Both upstream (5') and downstream (3') boundaries for the toxigenic element were examined in six toxigenic strains which vary considerably in toxigenicity to determine if there were variations among their respective toxigenic elements. The toxigenic element is highly conserved in these six strains. In the three nontoxigenic strains examined, a short fragment (127 bp) occupies the same chromosomal location as the large 19.6 kb toxigenic element.