Clostridium difficile was associated with five outbreaks of diarrhea occurring in three day-care centers caring for children less than 2 years of age during a three and one-half-month period; two centers had one outbreak of diarrhea, and one had three outbreaks. The frequency of isolation of C. difficile and its toxin was determined in stool specimens from 65 children attending the three day-care centers. Twelve of 21 (57%) children who had diarrhea excreted C. difficile and its toxin, whereas only four of 44 (9%) children who did not experience diarrhea yielded the organism and the toxin. Five of the 12 symptomatic children with C. difficile and its toxin had received prior antimicrobial therapy for upper respiratory tract infections. An eight-month prospective study was then carried out in one room of the center where the three outbreaks had occurred. Initially, one child had culture-positive stools for C. difficile. During the first 13-week study period six additional children of the 12 (50%) in the room under study became colonized with C. difficile. Six of the seven children who excreted C. difficile and its toxin experienced diarrhea with C. difficile toxin-positive stools by the end of the 13 weeks. Environmental contamination increased as more children became colonized and developed diarrhea. Electrophoretic evaluation of environmental and stool isolates obtained during one outbreak of diarrhea showed that the isolates had an identical protein pattern. Findings of this study indicate the importance of searching for C. difficile in children in day-care centers who develop diarrhea.