Escherichia coli strains causing human extra-intestinal infections may be divided into two groups, B1 and B2 according to the electrophoretic patterns of carboxylesterase B. This study compares the restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) for 45 B1 strains and 45 B2 strains to examine the genetic structure of B2 strains and to distinguish them from B1 strains. The isolates were chosen for diversity in their allozymes of esterases, B, A, C and I, their production of virulence factors (alpha-haemolysin, mannose resistant haemagglutinin and cytotoxic necrotizing factor) and certain O antigens, and their pathological and geographical origins. DNA was digested with HindIII and BamHI restriction enzymes and analysed by Southern blotting. The resulting rDNA RFLP patterns of B2 strains were distinct from those of the B1 strains. Moreover, the B2 strains appeared to be less heterogeneous than the B1 strains. The B2 strains gave 13 ribotypes (resulting from the combination of the rDNA RFLP patterns obtained with HindIII and BamHI digestions) while the B1 strains gave 32 ribotypes. Correspondence analysis of the data showed that several clusters of strains were identified in the B2 strains by particular ribotypes, certain associations of esterase B and A electrophoretic variants, O serotypes and virulence factor production. In contrast, these parameters appeared to be unrelated in the B1 strains, reflecting their heterogeneity. These findings, which differentiate two levels of genetic heterogeneity within E. coli pathogenic isolates, indicate that the B2 strains constitute a phylogenetically distinct group within the species.