A luteinizing hormone (LH) enriched fraction from the whole pituitary glands of buffaloes has been obtained by a simple, economical and scalable protocol using two gel filtration steps. An extract of pituitary glands was mixed with blue dextran and applied to the first Sephacryl S-200 gel filtration column. Blue dextran fractions eluting in the void volume of the column together with bound LH were pooled, reduced in volume and re-loaded on to the second gel filtration column, pre-equilibrated with 50 mM phosphate buffer pH 7.3 containing 1M KCl. In the presence of I M KC1, LH dissociated from the blue dextran and eluted in the same elution volume as in the case of dimeric native form of buffalo LH, while blue dextran eluted in the void volume of the column. The protein obtained from the peak in the second gel filtration was found to be highly immunorective against bovine LH beta subunit specific antiserum and it was 46-folds purer over the starting material as indicated by the direct binding ELISA. SDS-PAGE of the purified LH showed two major bands of LH, which was confirmed by western blot analysis. The yield of LH was found to be 262 mg LH/kg of whole pituitary glands. (c) 2005 Elsevier Ltd. All rights reserved.