Previously described GM1 ganglioside enzyme-linked immunosorbent assays (GM1-ELISA) for the detection of Escherichia coli heat-labile enterotoxin (LT) showed sensitivity equal to the Y-1 adrenal cell assay when anti-LT (a reagent not commercially available) was used. However, when antitoxin to immunologically related (commercially available) cholera toxin was substituted, a marked loss in sensitivity occurred. We modified the GM1-ELISA that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal cell assay. When five media commonly used for LT production were compared, Mundell's Casamino Acids medium was shown to be significantly superior. Lincomycin (45 micrograms/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polymyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved not only assay time, but also materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all three observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.