An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.