The 20-kDa growth hormone (GH) is generated from alternative splicing of the primary transcript of full-length 22-kDa GH. We have studied the regulation of 20-kDa GH over a range of pathophysiological conditions and in response to pharmacological stimulation using isoform-specific enzyme-linked immunosorbent assays (ELISAs). Mean 24-h levels of 20- and 22-kDa GH were higher in acromegaly and lower in GH deficiency than in normal subjects, with the 20-to-22-kDa ratio not different between the three groups. In normal subjects, 20-kDa GH was secreted in a pulsatile manner throughout the day, with peaks coinciding with those of 22-kDa GH. However, the half-life of 20-kDa GH (18.7 +/- 0.8 min) was significantly longer than that of 22-kDa GH (14.7 +/- 0.8 min; P < 0.02). Insulin-induced hypoglycemia, androgen, and oral estrogen caused a parallel and proportionate increase in both isoforms. Octreotide suppressed 20-kDa less rapidly than 22-kDa GH in blood. Administration of recombinant 22-kDa GH in normal subjects rapidly reduced the 20-kDa GH levels. In conclusion, 20-kDa GH is cosecreted with and circulates at a constant proportion of 22-kDa GH. The 20-kDa GH level is reduced by administration of exogenous 22-kDa GH, suggesting rapid negative feedback regulation on pituitary release.