We conducted a cross-sectional study of 79 children attending seven day care centers in Houston, Texas, to detect fecal gram-negative bacilli resistant to trimethoprim (TMPr) and ampicillin (AMPr). Fifteen children (19%) were colonized with TMPr Escherichia coli; all but one strain were also resistant to sulfonamides. Most of the children with TMPr E. coli were clustered in center A, where 11 (37%) of 30 children were colonized; only four (8%) of 49 children in the other six centers were colonized with TMPr E. coli (P less than .005). The TMPr E. coli isolates from 10 of the 11 children in Center A had a similar antibiogram, which included resistance to sulfonamides, ampicillin, and streptomycin; eight had a similar total plasmid pattern, an observation suggesting spread within the day care center. Children colonized with AMPr E. coli were present in all centers, although a higher percentage of children in center A were colonized than in the other centers combined (70% vs. 35%; P less than .01).