The development of a sensitive enzymeimmunoassay (EIA) for the determination of estrone (E1) and estradiol-17β (E2 β) in bovine plasma is described. The assay is a homologous double-antibody EIA with E2β 17hemisuccinate (HS) as hapten for the immunoreactive reagent. The antiserum was raised against E2β 17HS bovine serum albumin conjugate in the rabbit, and E2β 17HS-horseradish peroxidase was used as steroid-enzyme conjugate. Each estrogen EIA was distinguished only by using the each working standard and sample for the EIA. Bovine plasma E1 and E2β were extracted and purified before EIA. The antiserum was used at 1:1,750,000 dilutions for EIA. Estrone and E2β showed high cross-reactivity with the antiserum (E1: 350.7%, E2β:100%). The sensitivities were <0.03 pg/well for E1 and <0.12 pg/well for E2β. Recovery rates of E1 and E2β added to bovine blood plasma were 94.5% and 93.9%, respectively. The precision for EIA of estrogens was below 9.7%. The profiles of either estrogen as determined by EIA corresponded closely well with follicle dynamics in the cow during the estrous cycles and with placental function in pregnant animals. In conclusion, our new EIA can be applied with sufficient sensitivities, recovery and precision for the routine analysis of E1 and E2β concentrations in bovine plasma.