A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp(8)Arg and Ile(15)Thr) and an extra glycosylation consensus site (Asn(13)-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity approximately 12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS-PAGE. Although the mutations in V-LHbeta did not significantly affect the affinity of LH receptor (LHR) binding (Kd approximately 0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP(3)) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5-9 min) than that of WT-LH (12-22 min) and human chorionic gonadotropin (hCG; 50-70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man(5,8,9)) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHbeta allele.