A 12.3 kb DNA fragment encompassing the botulinum neurotoxin C1 (BoNT/C1) gene and an upstream flanking region was sequenced from Clostridium botulinum C 468 phage 1C. The resulting bont/C1 locus includes six genes which are organized into three transcriptional units. Cluster 1 encompasses the bont/C1 gene and an upstream gene encoding a non-toxic protein associated with the toxin (Antp139/C1). Transcriptional analysis revealed that these two genes form an operon; the bont/C1 gene can be transcribed alone or co-transcribed with antp139/C1. Cluster 2 encompasses three genes (antp33/C1, antp17/C1 and antp70/C1), which also form an operon. The corresponding proteins are similar to components of the hemagglutinin complex associated with BoNT/A and BoNT/B of C. botulinum A and B. In addition, Antp33/C1 is identical to HA-33, an hemagglutinin encoded by C. botulinum C-Stockholm phage C-St; Antp70/C1 displays some relatedness to C. perfringens enterotoxin. The third transcriptional unit consists of orf-22, which encodes a basic protein showing 29% identity with the gene product of uviA, a plasmid-encoded protein of 22 kDa which has been identified as a positive regulator of the bacteriocin production in C. perfringens. Orf-22 could be an effector controlling the expression of the bont/C1 and its antp genes in C. botulinum C 468.