The Mycobacterium paratuberculosis Antibody Test Kit® (IDEXX Laboratories, Westbrook, ME) was adapted for use with human sera by checkerboard titration of critical reagents. This bovine ELISA has proven effective in diagnosing paratuberculosis in cattle. Serum from a veterinarian who was twice accidentally inoculated with the vaccine for Johne's disease, was used as a positive control, and serum from a normal, healthy, non-veterinarian was used as a negative control. Optimal discrimination between positive and negative control sera was obtained using a serum dilution of 1:200 in absorbing diluent, an absorption time of 30 minutes, and a polyclonal HRP-conjugated goat anti-human conjugate at a dilution of 1:4000. Using these reagent concentrations, a kinetic ELISA determined the optimum substrate-enzyme reaction time to be 12 minutes. ELISA results were standardized by expression of each optical density value as a percentage of the controls (EV%); negative control=0%, positive control=100%. This prototype assay was used in a preliminary study to evaluate 2 populations of human sera for the presence of M. paratuberculosis antibody. One population consisted of 200 serum samples obtained from Red Cross blood donors in Dane Co., WI and the other population consisted of 300 serum samples obtained from Barron Co., WI dairy farm families. The Red Cross samples had EV% values ranging from 41%to 355% with a meanSD of 3.6%±48.8%. The Barron Co. samples had EV% values ranging from -17% to 633% with a mean±SD of 49.2%±99.1%. Statistical analysis (Mann-Whitney test) showed that the 2 populations were significantly different (P<0.0001). Results show that this ELISA may be useful in determining the prevalence of antibodies to M. paratuberculosis in human sera. This assay will be used to measure exposure rates in selected human populations considered at risk, and in Crohn's disease patients.