The specific identification of verocytotoxin (VT)-producing Escherichia coli (VTEC) requires the detection of VTs in bacterial culture filtrates or the detection of genes encoding these toxins in bacterial cells by specific DNA probes. The standard method for detecting these toxins involves a time-consuming, labor-intensive, and expensive cytotoxicity assay. We have developed a specific, highly sensitive receptor-specified enzyme-linked immunosorbent assay (RELISA) to detect VT1, one of at least two VTs implicated in human disease. The assay is based on the affinity of VT1 for the glycolipid globotriosyl ceramide (Gb3). Gb3 was de-N-acylated to yield lyso-Gb3, which is more polar but retains VT1 binding. Lyso-Gb3 was used to sensitize microdilution plates to bind VT1 for subsequent immunodetection. This RELISA was used to detect VT1 in the culture supernatant of a variety of bacteria of known VT status. The assay was compared with the highly sensitive cell cytotoxicity assay for their abilities to detect VT. The RELISA was as sensitive as the cytotoxicity assay and, in a blind study, 100% specific. This assay will provide a quick, specific, efficient adjunct to the diagnosis and epidemiological study of VTEC infections and their relationship to human disease.