Transesterification of soybean oil was carried out over calcium oxide supported on Al2O3 as solid base catalysts. Solid base catalysts were synthesized by wet impregnation of CaO on acidic, basic and neutral Al2O3. Solid basic catalysts investigated in this study were characterized by BET surface area, XRD, FTIR, CO2-TPD and TG/DTA techniques. Formation of fatty acid methyl esters (FAME) in this study was influenced by the nature of the catalyst prepared on different Al2O3 supports. CaO supported on neutral-Al2O3 exhibited higher FAME yield and the activation energy was determined to be 30.7 kJ/mol. The formation of the calcium diglyceroxide (CDG), on the surface of CaO supported on neutral Al2O3 was established by C-13 NMR spectroscopy. The catalytic system (with CaO + COG) on neutral Al2O3 showed higher activity in the transesterification of soybean oil. Among the catalysts studied the conversion of soybean oil to FAME formation was as follows: CaO/neutral-Al2O3 > CaO/basic-Al2O3 > CaO/acidic-Al2O3. The presence of COG on neutral Al2O3 probably contributes to the higher activity. To our knowledge, this is the first report on identifying the formation of CDG on neutral Al2O3 during the production of biodiesel. (C) 2012 Published by Elsevier B.V.