Hamster pituitaries were collected over an 8-yr period to obtain 3.5 g of dried pituitaries that were submitted to fractionation by extraction with 40% ethanol-acetate buffer at pH 5.5. The 80% acetone precipitate contained all of the gonadotropin activity. This enriched fraction was further purified by Sephadex G-100 and CM-Sephadex chromatography to obtain 4 mg purified hamster LH (haLH). From 1.5 mg haLH, 0.6 mg of the alpha- and beta-subunits were isolated by countercurrent distribution. haLH assayed 0.53 times NIH-LH-S19 by radioligand assay or 0.16 times NIH-LH-S19 by rat Leydig cell steroidogenesis assay. The amino acid N-terminal sequence was: haLH alpha, L-P-D-G-D-F-T-M-Q-G-C-P-; and haLH beta, S-R-G-P-L-R-P-L-C-R-P-I-N*-A-, as represented by the single letter code. The asparagine residue marked by an asterisk was placed by analogy, as this is apparently a carbohydrate-bearing residue. The amino acid compositions of haLH and its subunits are presented. Both subunits are glycoproteins.