Aside from the polysaccharide capsule, little is known about bacterial factors that contribute to the virulence of group B streptococci (GBS). In an effort to identify bacteria more likely to cause invasive disease, we determined GBS C5a-ase activity and HindIII-derived restriction digest patterns (RDPs) of chromosomal DNA from 41 invasive type III GBS strains isolated from sterile body sites (blood, CSF, or joint) and 29 colonizing strains isolated from vaginal secretions. The similarity between RDPs was expressed as a Pearson product moment coefficient that compares densitometric analyses of RDPs between strains (J. Med. Microbiol. 35: 297, 1991). Bacteria were grouped into one of four RDP types: III-1 (4.3%), III-2(25.7%), III-3 (58.6%) and III-4 (11.4%). Bacterial RDPs within each type were similar at a coefficient value ≥0.93. There was no obvious correlation between expression of C5a-ase and invasiveness in any of the RDP types. All type III-1 and III-2 strains tested expressed C5a-ase, while all of the type III-4 strains did not express C5a-ase activity. 66% of the III-3 strains expressed C5a-ase, but C5a-ase negative and positive strains were equally likely to be invasive isolates. Type III-3 strains that were very similar to each other by RDP typing (coefficient ≥0.99) were concordant for the presence or absence of C5a-ase, supporting the assertion that the RDP typing identifies closely related strains. Invasive isolates were significantly more likely to be type III-3 (78%) than were colonizing isolates (33%) (p<.001, Chi-squared). These data suggest that RDP type III-3 strains are more likely to cause invasive disease in human infants than other RDP types. Further characterization of these strains may help identify type III GBS virulence factors.