Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx_2d. The stx₂ operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx₂, stx_{2c vha}, stx_{2c vhb}, or stx_{2d EH250}. Subsequently, the stx_{2c vha} and stx_{2c vhb} operons were screened for the absence of a PstI site in the stx_2A subunit gene, a restriction site polymorphism which is a predictive indicator for the stx_2d (activatable) genotype. Twelve STEC isolates carrying putative stx_2d operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx_2d. The complete nucleotide sequences of seven representative stx_2d operons were determined. Shiga toxin expression in stx_2d isolates was confirmed by immunoblotting. stx_2d isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages φ1662a and φ1720a carrying the stx_2d operons. RFLP analysis of bacteriophage genomic DNA revealed that φ1662a and φ1720a were highly related to each other; however, the DNA sequences of these two stx_2d operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.