Ovine luteinizing hormone (oLH), a pituitary hormone with sulfated asparagine-linked oligosaccharides, was examined with regard to how its isoforms having different isoelectric points (pIs) modulate in vivo biological activity. oLH was separated into five fractions by means of an isoelectric focusing (IEF) column, i.e., IEF fr 1 (pI > 10.78), IEF fr 2 (pI 10.78-10.26), IEF fr 3 (pI 10.22-10.07), IEF fr 4 (pI 9.99-9.84), and IEF fr 5 (pI 9.76-9.48). Of these, we studied the three major fractions, IEF fr 2, 3 and 4. The binding of 125I-oLH to rat testis homogenates was inhibited most strongly by IEF fr 4, followed by 2 and 3. The ability to stimulate cyclic AMP release from dispersed rat Leydig cells followed the same sequence. After bolus injections of IEF fractions, the plasma immunoreactive oLH levels of male rabbits were measured. IEF fr 4 and 3 had longer plasma half-lives than 2. As expected, plasma testosterone levels after injections increased with the additive effects of in vitro activity and the rate of clearance from the circulation, i.e., IEF fr 4 > 3 = 2. In vivo renotropic activity, 3H-thymidine incorporation into the renal DNA of hypophysectomized, castrated rats, was found only in IEF fr 4. Our study indicates that an oLH isoform with a lower alkali pI had stronger LH receptor binding and in vitro bioactivity and a longer plasma half-life, resulting in stronger in vivo bioactivity.