A combined physical and genetic map of the genome of strain SG24 of Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI. The genome of SG24 is circular, and the genome size is about 3.57 Mb. The linkages between 47 NotI and 32 SfiI fragments of V. cholerae SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine CeuI fragments were determined primarily by analyses of partial fragments of the CeuI-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a V. cholerae genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.