A method of estimating progesterone in buffalo whole milk by EIA using progesterone 6 beta-OH-hemisuccinate-horseradish peroxidase as the enzyme label and an antiserum raised against progesterone-7 alpha-carboxyethyl-thioether-BSA was developed. The microtitration plates used in the assay were first coated with affinity purified sheep IgG developed against rabbit IgG. The immune reaction was performed by incubating a mixture of 1 microliter of whole milk (diluted to 20 microliters with assay buffer), 100 microliters of enzyme label and 100 microliters of antiserum for 90 min in the dark. After washing the plates, 150 microliters of the substrate solution was added. The mixture was incubated in the dark for 40 min before the reaction was stopped and the optical density was measured at 450 nm. The calibration curve was sensitive in the range 0.8-40 pg/well, corresponding to 0.8-40 ng/ml. Milk samples from cycling buffaloes were tested for progesterone concentration by running parallel EIA and RIA. A good correlation of 0.91 was obtained and the estimated values were similar using both techniques. The method has demonstrated about 10 times greater sensitivity than RIA in buffalo milk.