Although the suckling mouse assay is widely used for the detection of heat-stable Escherichia coli enterotoxin (ST), few data have been published concerning the reproducibility, optimal growth, and test conditions of this assay. Four strains of toxigenic E. coli known to elaborate both heat-labile enterotoxin and ST or ST alone were used to study these parameters. ST activity after heat treatment and the effect of purified choleragen were also examined. ST production was optimal in Casamino Acids-yeast extract media, but both Trypticase soy and brain heart infusion broths resulted in several false negative reactions. Growing cultures in roller tubes was the most reliable method of ST production. Shaking-flask cultures and stationary-grown cultures resulted in suboptimal ST production in several strains. Optimal mouse incubation time was 3 h, and fluid secretion did not rise thereafter. Adequate toxin production occurred after 16 to 24 h of incubation. The coefficient of variation of various toxins tested on many occasions varied between 10.5 and 15.7%. Toxin activity was stable for 6 months when frozen at - 20 C. There was no decrease in ST activity when heated at 65 C for 15 min, but a small decrease was observed in two of four strains after heating at 100 C for 30 min. Choleragen, tested at various doses and at multiple times, gave uniformly negative results. These studies indicate that when done under the proper conditions, the suckling mouse assay is a simple, rapid, and reproducible assay for E. coli ST.