Serum fractions from normal subjects obtained by gel chromatography were investigated using 3 different assay systems: radioimmunoassay (RIA), radioligand receptor assay (RRA), and testosterone production assay (TPA). The bulk of immunoassayable and bioassayable LH[luteinizing hormone]-activity was found in 2 fractions differing widely in their molecular size. The slower moving component, designated as little LH, migrated identical to the radioiodinated pituitary hormone (LER 960 [LH reference preparation]) with a MW of about 30,000, while big LH appeared in an elution volume consistent with a MW range between 140,000 and 180,000. Concordance was seen between the LH-activities measured in all 3 assay systems. The RRA/RIA ratio varied between 1.6 and 8.9; the RRA/TPA ratio was close to unity. Treatment with 6 M urea and 0.1% mercaptoethanol and exposure to different pH values and salt concentrations did not change the elution position of the 2 LH components. Big and little LH appeared unaltered after refiltration, and no conversion to each other could be found. In another experiment, injection of gonadotropin releasing hormone (Gn-RH) into a male induced a profound shift of LH towards the low molecular weight species. Kinetic uptake studies with big and little LH using RRA showed identical affinities to the receptor preparation. Ion exchange chromatography of serum, however, did not give 2 LH components, indicating no major differences in charge properties. This finding could be confirmed by preparative gel isoelectric focusing. The RRA potencies following gel filtration were in good agreement with that applied to the column, however, the immunological activities exceeded that of loaded by a factor 3-4. A new aspect of serum LH heterogeneity is the finding of a low molecular substance (MW approximately 1000) in the outer dialysate of serum, which has LH like activity in all 3 assay systems.