The 20K hGH isoform is produced by alternative splicing of GH mRNA, and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunoadsorbent assays (ELISAs) specific to 20K and 22K hGH. The serum levels of 20K hGH after overnight fasting was 118±178pg/mL (N=282) in normal women, significantly higher than in normal men (64±170pg/mL, N=226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 ±2.6%, N=176) and women (6.3±2.1 0, N=263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P<0.001) higher in patients with active acromegaly (9.2±2.2%, N=33) and in patients with anorexia nervosa (9.0±1.9, N=8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of the 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or chronic excess of 22K hGH altering the metabolic clearance rate of 20K hGH. The values in patients with adult growth hormone deficiency (GHD), hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests such as insulin tolerance test and GRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH.