During a period when certain neonates in our nursery developed necrotizing enterocolitis, we studied stool specimens from a population of symptomatic and asymptomatic patients for the presence of Clostridium difficile and its toxin. The presence of the organism among nursery personnel and in the nursery environment was also evaluated. Results showed that five symptomatic neonates and 17 asymptomatic neonates in a population of 37 patients studied in our neonatal intensive care and intermediate care nurseries had positive stool cultures for C. difficile. Toxin was detected in stool from four of five symptomatic patients and was present at dilutions of greater than 1/125 in stool from six asymptomatic patients who were culture positive for C difficile. The organism was not found in stool samples from any nursery personnel but was recovered from the hand culture of a nursery worker and from the inanimate environment. Although our findings support the notion of nosocomial spread of C difficile, we were unable to clearly implicate the organism as the cause of necrotizing enterocolitis in our nursery.