In the neonatal intensive care unit (NICU) of one hospital, 16 infants became colonized or infected with multiply-resistant Escherichia coli (MR-E.coli) over an 8-month period. Isolates were obtained from blood, urine, and sputum of three patients and from rectal surveillance cultures of 13 patients. The one patient with the blood isolate died. A matched case-control study identified continuous feeding (nine of 16 cases vs. one of 16 controls, p ≤ 0.001) and receipt of aminoglycosides (p ≤ 0.03) as risk factors. For case-babies not exposed to continuous feeding, duration of bolus feeding was significantly greater than for their controls (cases, 22 days; controls, 7 days; p ≤ 0.02). All 16 isolates were the same serotype and were resistant to amikacin, tobramycin, kanamycin, and gentamicin. The epidemiologic investigation suggested that MR-E. coli may have spread from person-to-person on the hands of personnel and that MR-E. coli persisted in the NICU for 8 months until effective control measures were instituted.