Coagulase-negative staphylococci are now the major cause of bacteremia in neonatal intensive care units. To date, coagulase-negative staphylococci causing neonatal infections have been found to be distinct when typed by standard techniques. To determine whether or not an endemic strain could be identified using more discriminatory techniques, we characterized coagulase-negative staphylococci isolates obtained from a prospective study of coagulase-negative staphylococci bacteremia in a neonatal intensive care unit during 1984 through 1985, by standard techniques supplemented with DNA-DNA hybridization and restriction endonuclease analysis. We typed 58 strains that were isolated from 52 episodes of bacteremia in 38 neonates. There were 46 isolates of Staphylococcus epidermidis. Three pairs of strains were identical, and each strain was from a different patient. There were 12 isolates of Staphylococcus haemolyticus. Ten were identical, referred to as strain TOR-35, and had been isolated from eight different infants. Characterization of strains obtained in 1986 from a prospective study of coagulase-negative staphylococci-colonizing neonates admitted to the same neonatal intensive care unit found the TOR-35 strain had colonized 6 of 17 neonates by day seven. A point prevalence survey of all neonates in the same neonatal intensive care unit in 1990 found 5 of 30 neonates to be colonized with the TOR-35 strain. Therefore, we were able to identify an endemic strain of S haemolyticus that caused multiple episodes of bacteremia during a 6-month period and remained present in the same environment for a 5-year period.