Serum levels of total insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) reflect endogenous GH secretion in healthy children, which makes them good diagnostic markers for screening of GH deficiency (GHD) in short children, although some controversy still exists. Only a minor fraction of the total IGF-I circulates in its free form, which is believed to be the biologically active form. However, our knowledge of the clinical or physiological value of determination of free IGF-I in serum is limited at present. In adults, the diagnostic value of total IGF-I and IGFBP-3 determinations in patients suspected of GHD has only been reported in a few studies, whereas no previous reports on the diagnostic value of free IGF-I levels in adults suspected of GHD exist. Serum levels of free IGF-I were determined in 1430 healthy children, adolescents, and adults by a newly developed, commercially available immunoradiometric assay (Diagnostic Systems Laboratories) to establish valid normative data for this analysis. We studied the diagnostic value of free IGF-I in relation to total IGF-I and IGFBP-3 determinations in adults who were suspected of GHD. A GH provocative test, using oral clonidine, was performed in 108 adult patients who had previously been treated with GH in childhood. In healthy subjects, free IGF-I levels increased during childhood, with the highest mean values during puberty. After puberty, a subsequent decline in serum levels of free IGF-I was apparent. We found, unmeasurable free IGF-I values in 34 of the prepubertal children (3.3%). All individuals over 8 yr of age had measurable free IGF-I levels that amounted to approximately 1% of the total IGF-I concentrations. Free IGF-I levels were below--2 SD in 56 of 79 GHD patients (sensitivity, 71%) and above--2 SD in 24 of 29 patients with a normal GH response (specificity, 83%). Multiple linear regression analysis demonstrated that free IGF-I was significantly dependent on peak GH levels, duration of the disease, and number of other pituitary axes affected. We conclude that free IGF-I serum levels increase during childhood with a peak in puberty, whereafter free IGF-I levels return to prepubertal levels. Three percent of healthy prepubertal children had unmeasurable free IGF-I levels using this assay. We found that determination of the free IGF-I serum concentration may predict the outcome of a GH provocative test in adults suspected of GHD, but that a single determination of free IGF-I offered no significant advantage compared to determination of total IGF-I or IGFBP-3 serum levels.