This experiment was carried out to determine the progesterone concentration of bovine plasma by liquid phase double antibody enzyme immunoassay. The optimum conditions of assay-system, enzyme conjugate and gelatin were investigated. The sensitivity, recovery rate and reproducibility by this assay were also analyzed. The suitable supplementation level of gelatin was 0.2 %. As the gelatin level increased to 1 %, coefficient variation of interassay was shown to be irregular. The optimum dilution rate of enzyme conjugate was 30 times. Enzyme activity was greatly fluctuated depending on the minor condition of enzyme conjugate technique. Therefore, it was considered to be checked when determined. The sensitivity of the assay was 12 pg/tube. Recovery rate was decreased when the amount of sample was too little or too much, but the recovery rate was high as 97.8 % when the amount of sample was between 50 and 200 micro l. Mean intra-assay and inter-assay coefficient of variation was 4.5 % and 5.9 %, respectively. By using liquid phase double antibody enzyme immunoassay, progesterone in plasma can be detected, and also this assay system is applicable to study the physiological function of progesterone and diagnosis of reproductive disorders