An enzyme-linked immunosorbent assay (ELISA) consisting of a double sandwich technique with rabbit and sheep antibodies, was developed for the detection of cytotoxic necrotising factor (CNF) in extracts of Escherichia coli strains. The assay was evaluated by comparison with the results obtained with an assay based on toxicity for HeLa cell cultures. In a study of extracts of 27 CNF+ and 45 CNF- strains obtained by ultrasonic disintegration, no false positive and only three false negative results were recorded; the latter were obtained with strains that produced less CNF than any of the others examined. Frozen-thawed extracts contained about four times more CNF cytotoxic activity than extracts prepared ultrasonically; the testing of 54 CNF+ and 68 CNF- frozen-thawed extracts resulted in no false positive and only one false negative response. Whichever type of extract was used, no significant cross-reaction was observed with heat-labile (LT) or heat stable (ST) enterotoxin, verotoxin (VT1, VT2), haemolysin, or Vir cytotoxin.