To follow the emergence of surface colonization with coagulase-negative staphylococci in neonates, we sampled four surface sites (axilla, ear, nasopharynx, and rectum) in 18 premature infants during the first 4 weeks of life. Swabs were obtained on the first day of life, twice weekly for 2 weeks, and weekly thereafter. Isolates were characterized by species, biotype, antibiotic susceptibility patterns, and slime production. Over 4 weeks the percentage of infants with Staphylococcus epidermidis as the only surface coagulase-negative staphylococci rose from 11% to 100%. Predominance of a single S. epidermidis biotype increased from none to 89%. Multiple antibiotic resistance rose from 32% to 82% of isolates, and the prevalence of slime production increased from 68% to 95%. This microbiologic pattern was established by the end of the first week of life and persisted throughout the month of study. In three infants, S. epidermidis sepsis developed with organisms identical to their predominant surface isolate. We conclude that species, multiple antibiotic resistance, and slime production appear to confer a selective advantage for the surface colonization of premature newborn infants in the intensive care nursery environment. Infants so colonized may be at greater risk for subsequent infection with these strains of coagulase-negative staphylococci.