A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed. The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by S1 nuclease. Cleavage of the chromosome with PacI and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb. Digestion of the chromosome with I-CeuI produced 10 fragments, indicating that 10 rrl (23S) genes were likely to be present. Hybridizations between DNA fragments generated with PacI, PmeI and I-CeuI were used to initially determine the relationship between these segments. To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for PacI and PmeI, producing pJP9540. Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, PacI and PmeI cleavage determined the location of genes within macrorestriction fragments with the additional bands produced forming hybridization probes. From the data generated, it was possible to form a physical map comprising all the fragments produced by PacI and PmeI, and assign the contig of I-CeuI fragments on this map. The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data.