In order to elucidate some of the factors responsible for the high rate of nosocomial infection associated with neonatal intensive care, we studied bacterial colonization in 63 infants admitted to a neonatal intensive care unit. In a six-month period, cultures of nose, throat, umbilicus, and stool were obtained on admission and every three days from all infants staying in the NICU greater than or equal to 3 days. Study infants did not develop "normal" aerobic flora. Forty-eight percent of infants grew Escherichia coli from stool, but 52% had stool colonization with Klebsiella, Enterobacter, or Citrobacter, the only other Enterobacteriaceae encountered. KEC were also isolated from throat, nose, and umbilicus in 22%, 22%, and 24% of patients, respectively. The risk of stool colonization with KEC increased with duration of hospitalization: 2% of infants were colonized on admission, 60% after 15 days, and 91% after 30 days. Stool colonization with E. coli seemed to protect infants from colonization with other gram-negative bacilli. Thirteen of 20 infants, however, developed pharyngeal GNB colonization in spite of pre-existing abundant growth of alpha streptococci. Antibiotic therapy for greater than 3 days was associated with the isolation of KEC in stool and GNB in the throat, but birth weight less than 2,500 gm and lack of breast milk feedings were not.