Human GH (hGH) consists of several molecular forms. Monomeric forms present in pituitary extracts include the single chain, 22,000 mol wt form (22K; hGH-B); a 20,000 mol wt, single chain variant (20K); three proteolytically cleaved, two-chain forms (hGH-C, -D, and -E), acetylated, deaminated, and slow-migrating forms. It is not known which of these forms are secreted in vivo or whether peripheral organs contribute to the interconversion between some of these hGH forms. To answer these questions, we studied the molecular forms of hGH excreted in urine from normal volunteers and from an acromegalic patient, as urinary hGH presumably reflects integrated plasma hGH. hGH was extracted from urine by hollow fiber diafiltration and concentration, followed by immunoadsorbent chromatography. The extracted hGH was examined by polyacrylamide gel electrophoresis under native as well as denaturing and reducing conditions and by isoelectric focusing. The predominant form of hGH in both normal urine and urine from the acromegalic patient was 22K, with small quantities (approximately 10%) of 20K and an unidentified acidic form also present. Cleaved forms with enhanced bioactivity (hGH-D and -E) and big hGH forms were not detectable. We conclude that 1) the pattern of urinary hGH suggests that spontaneously secreted and circulating hGH is composed of at least three hGH forms, with 22K predominating; 2) the pattern of urinary hGH is similar to that of plasma hGH after L-dopa stimulation; 3) hGH excreted in acromegaly is indistinguishable from hGH excreted by normal subjects; 4) only a minute fraction (less than 0.01%) of the hGH secreted reaches the final urine; and 5) renal interconversion among hGH forms does not appear quantitatively important.