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Escherichia coli K1, a normal inhabitant of the human flora, is also an important cause of serious infections in newborns. We compared two molecular methods, ribotyping and arbitrarily primed polymerase chain reaction (AP-PCR), to study the apparent nosocomial transmission of an E. coli K1 clone in a nursery for premature neonates. Sixty-two E. coli K1 strains isolated from 41 premature neonates from December 1991 to June 1992 and six strains isolated from ambient sources were studied. Eight E. coli K1 strains isolated from infants in the nursery between 1989 and September 1991 were included as controls. The properties of the strains isolated between December 1991 and June 1992 were as follows: 43 belonged to ribotype I, 12 belonged to ribotype III, and the remaining 13 isolates were distributed among 10 ribotypes. The eight control strains belonged to seven different ribotypes, but none was ribotype I. Between December 1991 and February 1992, the majority of strains from premature infants colonized with E. coli K1 were of ribotype I. Isolates from the ventilation system and from a storage shelf were also of ribotype I. When DNA from 56 selected strains was tested by AP-PCR by using the 5'-TTGTAAAACGACGGCCAG-3' oligonucleotide, 15 different profiles were obtained. Twenty-five of 56 strains were of ribotype I and had identical profiles by AP-PCR. Strains with ribotypes VI, VII, and X to XV had different profiles by AP-PCR. We conclude that ribotyping and AP-PCR correlate well and permit demonstration of the nosocomial dissemination of E. coli K1 in a unit for premature neonates.