A sensitive method for the detection and analysis of Gram-negative bacterial lipopolysaccharide (LPS, i.e. endotoxin) blotted on membranes is described. LPSs were separated by SDS-PAGE and then transferred electrophoretically onto membranes. The LPSs on membranes were oxidized with periodate to create functional aldehyde groups, reacted with a hydrazide linked to a steroid, and visualized by an enzyme-labeled antibodies to the steroid in the presence of chromogenic substrate. Both nylon and polyvinylidene difluoride (PVDF) membranes provided high sensitivity in the detection of LPSs. However, proteins on the PVDF membrane were also stained. The resolution of banding patterns of LPSs from over 15 different bacterial species was similar to that of silver stained companion gels. The detection of smooth LPS from Pseudomonas aeruginosa F-D type 1 and rough LPS from Escherichia coli K12 was sensitive to 10-20 nanograms.