The ability to attach to epithelial cells, efface the microvillus surface, and disrupt the underlying cytoskeleton is characteristic of enteropathogenic Escherichia coli (EPEC). Recently, eae, a gene necessary for this phenomenon, was described (A. E. Jerse, J. Yu, B. D. Tall, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 87:7839-7843, 1990). We report the use of a novel suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis to construct an eae deletion mutant of EPEC. This system enables positive selection for the loss of vector sequences. The resulting mutant, CVD206, is indistinguishable from the wild-type strain except for the loss of a 94-kDa outer membrane protein and attaching and effacing ability. Both the 94-kDa outer membrane protein and attaching and effacing ability are restored upon reintroduction of the eae gene on a plasmid. These results confirm the role of the eae gene in the attaching and effacing activity of EPEC and establish the utility of a new system for the construction of deletion mutations.