Abstract A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and E1 Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role