Purified heat-stable enterotoxin (ST) prepared from supernatants of Escherichia coli strain 53402 A-1, isolated from a patient with diarrhea, caused an increase in the permeability of the small blood vessels of the skin of adult rabbits after intracutaneous injection. Increased permeability was manifested by localized accumulation of intravenously injected blue dye at the injection sites. Permeability factor (PF) activity reached a peak 1 h after injection, and recovery of normal permeability was nearly complete in 3 h. Residual PF activity had disappeared by 24 h. Dose-response curves demonstrated a straight-line relationship between the logarithm of the dose and mean blueing diameter over a range of 3 to 12 mm. The PF assay was less sensitive that the suckling mouse assay, and adult rabbits varied in their sensitivity to the PF effect. Five to 100 mouse units of ST were required to consistently evoke strong and unequivocal blue lesions of 7 mm or more in diameter. PF activity was reduced 50 to 70% by heating at 70 degrees C for 30 min, 60 to 80% by boiling for 30 min, and 95 to 98% by autoclaving for 15 min, but it was unaffected by treatment with cholera antitoxin. These findings are consistent with the notion that PF activity is a property of the ST molecule itself. This PF assay cannot be used for the detection of ST in crude culture supernatant fluid because of low sensitivity and the presence of nonspecific PF effects of culture media and other E. coli products.