Escherichia coli K1 is the most common cause of gram-negative neonatal bacterial meningitis and septicemia. In an attempt to identify genetic markers in E. coli K1 that are associated with the capacity of the organism to cause neonatal meningitis, we used rRNA gene restriction patterns. E. coli strains isolated from the CSF of neonates with meningitis (n = 43) on two continents were compared to strains isolated from the blood of neonates with bacteremia who did not have meningitis (n = 29) and to isolates from the vaginas of asymptomatic pregnant women whose neonates remained without infection (n = 39). E. coli strains from CSF are genetically less heterogeneous than isolates from blood and the vagina: 44.2% of the CSF isolates belonged to only two types, whereas no more than two blood vaginal strains were of the same type. After HindIII digestion, a 14.9-kb rDNA-containing fragment was found in 81.3% of the strains from CSF vs. 28.0% of the isolates from blood and only 12.8% of the vaginal isolates (P = .001). Thus, genotyping might provide markers to identify organisms in the maternal vaginal flora that are highly likely to cause neonatal meningitis. This observation may have very practical implications for the early identification of these organisms in pregnant women and thus for the selective establishment of preventive measures per partum or for the early treatment of colonized neonates.