A library of total Clostridium novyi DNA was established and screened for the -toxin gene (tcn) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcn fragments were isolated and subsequently the total sequence of tcn was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M_r 250 166 and pI 5.9. The N-terminal -toxin (Tcn) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcn and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcn constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19–52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.