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This study was designed ability using antibody against buffalo PAG to detect concentration of pregnancy-associated glycoprotein (PAG) in bovine plasma samples. Successful purification of buffalo PAG molecules with high enrichment degree by using Vicia villosa aûnity chromatography was recently reported. In this research, the RIA system using novel anti buffalo-PAG was used for bovine pregnancy diagnosis. In a total 437 blood sampling of dairy cow in Belgium were used. Three PAG-RIA systems were undertaken to measure PAG concentration in bovine routine blood samples: AS#497 (or RIA-1) using rabbit antisera against purified bovine PAG67kDa, AS#706 (or RIA-2) using rabbit antisera against caprine PAG55+62kDa and new AS#859 (or RIA-3) using rabbit antisera against buffalo PAG59.5-75.8kDa). The measured PAG concentrations were similar for the systems As#497 and As#859; although the dilution was shown parallel, the measured PAG concentrations were significantly different between systems As#706 and As#859. On the other hand, the coefficients of correlations between concentrations obtained by the use of As#859 and As#497 were highly comparable to those between As#859 and As#706 or As#497 and As#706. In conclusion, the present study clearly shows that antisera against buffalo PAG can be used to develop new PAG-RIA system for bovine pregnancy diagnosis with high comparable results.