An enzyme-linked immunosorbent assay for the quantitative detection of mucins secreted in culture by a human colonic epithelial cell line has been developed. Dilutions of mucin standards and culture media were applied to nitrocellulose membranes by vacuum filtration through a specially designed immunofiltration manifold. Sequential incubation of the nitrocellulose with blocking buffer, anti-mucin polyclonal antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG permitted quantitation of mucins. Linear and reproducible standard curves were obtained with mucins purified by gel filtration chromatography using a gel of large pore size. The assay was found to be simple, sensitive, reproducible, rapid and not influenced by contaminating proteins up to a concentration of 500 micrograms/ml. This assay has been used to monitor column fractions obtained during the analysis of mucin preparations, and to study the effects of cholera toxin on the secretion of mucins by cultured cells.