With regard to the assay of heat-stable enterotoxin (ST) from Yersinia enterocolitica, we made a comparative study of the conventional infant mouse test read at 4 h and a modified infant mouse test read at 2 h. The influence of several factors, such as the medium used to prepare ST, lysing of bacterial cells from the broth cultures used to prepare ST, and the temperature at which the inoculated mice were kept during the test, was also investigated. Thus, with a few exceptions, Pai-Mors medium was more suitable than Casamino Acids-yeast extract medium, for the preparation of yersinial ST. Gut/carcass weight ratios obtained with lysed supernatants or with supernatants from whole cultures of Y. enterocolitica were similar, suggesting that most of the ST produced by this microorganism in broth cultures is extracellular. The amount of ST produced by Y. enterocolitica, as well as the ambient temperature at which inoculated mice were kept during the assay, was found to influence gut/carcass weight ratios obtained with both tests. Enterotoxigenicity and the temperature at which mice were kept were interrelated, such that for weakly enterotoxigenic strains there were no significant differences among gut/carcass weight ratios for the conventional and modified infant mouse tests carried out at 18 or 25 degrees C, but at 30 degrees C the values in the modified test were higher for most ST preparations with Pai-Mors medium. The influence of ambient temperature was more pronounced at 37 degrees C, since most strains produced negative results in the conventional test at this temperature. We conclude that the conventional infant mouse test is adequate for assaying yersinial ST, provided that the temperature at which mice are kept during the assay is fixed at around 25 degrees C.