We have developed a rapid, simple and highly sensitive 'sandwich' enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor beta) in serum. The assay, performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor alpha does not cross-react even at 10(7)-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the quantification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.