Twenty kilodalton human growth hormone (20K-hGH) is a naturally occurring isoform lacking amino acid residues 32-46 of 22K-hGH. Due to this structural similarity to 22K-hGH, no one has constructed a specific and sensitive assay system for 20K-hGH, which can be used for measuring physiological concentration of this isoform in the circulation. To construct such a specific assay system, we have generated polyclonal antibodies (pAb) and monoclonal antibodies (mAbs) against recombinant 20K-hGH. Binding characteristics to 20K-, 22K-hGH and monkey GH (mGH) of these five mAbs, designated A23, B13, C02, D05, and D14, were analyzed by enzyme immunoassays (EIAs) and surface plasmon resonance analysis. Only one of them, the mAb D05, does not crossreact with 22K-hGH. It was also observed that mAb B13 does not crossreact with mGH, although the later is 96% homologous to hGH. Using these antibodies we have established several sandwich EIA systems for circulating 20K-hGH. The combination of A23 as a solid-phase antibody and B13 as a labeled antibody permitted both high sensitivity to 20K-hGH (< 0.1 ng/ml) and low cross-reactivities with 22K-hGH (< 2%), mGH (< 0.3%) and rat GH (< 0.1%). The clearances of administered 20K-hGH were determined by this combination in both rats and monkeys. In the assay of physiologically circulating 20K-hGH in humans, the combination of D05 and affinity-purified anti-20K-hGH pAb showed the highest sensitivity to 20K-hGH (< 10 pg/ml) and substantially no cross-reactivity with 22K-hGH (< 0.1%). The plasma 20K-hGH concentration in healthy female subjects was determined by this combination. The assay systems constructed here enables us to directly measure circulating 20K-hGH in physiological condition with no interference of 22K-hGH for the first time.