The products of the ipaB, ipaC, and ipaD genes are involved in the expression of the invasive phenotype in all species of Shigella and enteroinvasive Escherichia coli (EIEC). DNA probes derived from these genes are accurate indicators of the invasive phenotype (M. Venkatesan, J. M. Buysse, E. V. Vandendries, and D. J. Kopecko, J. Clin. Microbiol. 26:261-266, 1988); however, spontaneous loss of the invasion plasmid or selective deletion of invasion-associated genes may restrict the usefulness of such probes as general diagnostic tools. In this study, we report that laboratory-passaged strains of Shigella spp. and EIEC that were invasion and Sereny test negative were unable to hybridize to the ipaC DNA probe. However, a second DNA probe, derived from the Shigella flexneri ipaH gene, a multiple-copy element found on the chromosome and invasion plasmid that encodes a 60-kilodalton antigen, was more sensitive in its ability to detect virulent as well as avirulent shigellae and EIEC. Analysis of colony blots and stool blots from pediatric patients with diarrhea indicated that the ipaH probe was more effective in detecting shigellae and EIEC than was either the ipaC or 17-kilobase EcoRI fragment probe.