The insulin-like growth factor binding proteins (IGFBPs) are the carriers for insulin-like growth factor (IGF0-I and IGF-II. IGFBP-3 is GH-dependent and circulates associated with IGFs and an acid-labile subunit to form a 150-kilodalton (kDa) complex. In human serum, two immunoreactive molecular weight forms of IGFBP-3 have been identified. In human urine, radioimmunoassayable levels of IGFBP-3 have been detected. The objectives of this study were to characterize the molecular weight forms of IGFBP-3 in urine and serum of healthy children and adults and in children with GH deficiency (GHD), to quantify the urinary molecular weight forms of IGFBP-3, and to evaluate the relationship of these forms with GH therapy. Urine and serum were obtained from 12 prepubertal children with GHD, before and after 6 months of GH therapy, from 30 prepubertal healthy children, and from 8 healthy adults. Western immunoblotting (WIB) with IGFBP-3 antiserum (alpha IG-FBP-3g1) showed that in urine the most representative IGFBP-3 was a 17.7-kDa form. The 17.7-kDa IGFBP-3 was high in urine of healthy children compared with healthy adults and was low in children with GHD but increased after GH therapy. Urinary IGFBP-3 immunoreactive profile was determined by neutral-size exclusion chromatography, followed by IGFBP-3 RIA analysis of the fractions. Urine showed a major peak of IGFBP-3 immunoreactivity around 17 kDa. The 17-kDa urinary IGFBP-3 chromatographic peak averaged 8461 +/- 367 ng/12 h.m2 of body surface in healthy children, 3415 +/- 739 in adults (P < 0.001), 2294 +/- 354 in children with GHD before GH therapy (P < 0.001), and 7940 +/- 1874 in children with GHD after GH therapy. Urinary IGFBP-3 was also measured by RIA in unfractionated urine; healthy children showed levels significantly higher (14575 +/- 460 ng/12 h.m2) than adults (7823 +/- 1083, P < 0.001) and higher than children with GHD before GH therapy (4710 +/- 703, P < 0.001). Again, however, immunoreactive IGFBP-3 increased after GH treatment (12294 +/- 3394). In the serum of the healthy children we characterized by specific IGFBP-3 WIB analysis, a 17.7-kDa immunoreactive form of IGFBP-3 that was absent in the serum of healthy adults and low in patients with GHD, increased during GH therapy. Serum samples were subjected to neutral-size exclusion chromatography and the fractions were analyzed by WIB.(ABSTRACT TRUNCATED AT 400 WORDS)