Enterotoxin preparations derived from Escherichia coli strain H-10407 were shown to contain vascular permeability factor (PF) activity as well as diarrheagenic activity. Intradermal injection of E. coli enterotoxin (ECT) caused localized induration and permeability of small blood vessels of the skin to intravenously administered Evans blue dye. The PF assay described here demonstrated a linear dose response and was at least as sensitive as the adult rabbit ileal loop assay for detecting ECT. E. coli PF activity was heat labile and was neutralized by homologous antiserum. PF production was enhanced by the addition of yeast extract (up to 0.6%) to a Casamino Acids-salts medium. PF activity was detectable as early as 6 h in aerated (shake) cultures in the Casamino Acids-yeast extract-salts medium, pH 8.5, maximal at 18 h and essentially unchanged at 48 h. The skin test (PF) assay for ECT has numerous advantages over current assay methods which involve gastrointestinal challenge of experimental animals.