CONTEXT Approximately 50% of circulating GH is bound to the high-affinity GH-binding protein (GHBP), which is known to affect the pharmacokinetics, bioactivity, and quantitative determination of GH. Nevertheless, the presence of GHBP is rarely taken into account in the clinical use of GH measurements. OBJECTIVE Our objective was to develop an assay for free GH in serum. METHODS We used ultrafiltration by centrifugation. Due to the small molecular difference between GH and GHBP, the size of GHBP and GHBP-GH complexes was increased by preincubation of serum with a monoclonal GHBP antibody (MAb 263). RESULTS The ultrafiltration membrane almost completely retained all GHBP (>98.5%) and allowed free passage of unbound GH (>98.4%). Addition of increasing concentrations of GHBP reduced free GH dose dependently, and measured and calculated levels of free GH changed in parallel. During an insulin-tolerance test, free and total GH changed in parallel in all individuals (n = 11) and their peak values as well as area under the curve values were positively correlated (r = 0.89; P < 0.001 and r = 0.92; P < 0.001, respectively). Of note, the relative levels of free GH (calculated as the area under the curve of free to total GH) was inversely correlated with GHBP (r = -0.94; P < 0.001). CONCLUSION It is possible to measure free GH in human serum. Free GH correlated positively with total GH and inversely with GHBP. Measurement of free GH may be a helpful future tool in the management of GH disorders and in studies of GH-GHBP interrelationships.