Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 10(3) gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 10(10) GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 10(5) GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 10(4) GC per g of sample). The stx(2) genes and stx(2) variants were detected in the viral fraction of some of the samples after sequencing of stx(2) fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.