Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of 2-day-old Swiss Webster suckling mice were used. For this study, 5, 10, 25 and 50 micrograms of insulin was given orally to half the mice in each group for 7 days. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, a suckling mouse assay in which 1 microgram of STa was given to all mice in insulin-treated and control groups was performed. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin-treated groups compared to control mice (P < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an up-regulatory effect on the STa-receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction, which is a major cause of diarrhea in newborn animals and human infants.