The binding in vitro of 125I-human or bovine growth hormone (GH) to normal female rabbit serum has been studied using gel filtration to separate bound and free hormone. On Ultrogel AcA34 columns, a substantial peak of specific 125I-GH binding was observed at a MW approximately 120 000. This peak was not precipitable by 12.5% polyethylene glycol, a method used widely for solubilized hormone receptors. Assuming a 1:1 binding stoichiometry between GH (MW 21 000) and the binding protein, the MW of the binding protein would be approximately 100 000. Gel filtration of serum alone, followed by assessment of 125I-hGH binding in column fractions, indicated the binding protein had a similar MW (83 000-107 000). Specific binding of 125I-hGH was dependent on incubation time (equilibrium being reached in 2 h at 21 degrees C), Ca2+ concentration (0.5-2.0 mM) and serum concentration (a 1:5 dilution of serum giving 45.2 +/- 1.7% specific binding; mean +/- SE, n = 10). Binding was completely reversible (t 1/2 approximately 1.5 h) and specific for somatotrophic but not lactogenic hormones. Scatchard analysis revealed linear plots with a Ka 1.59 +/- 0.11 X 10(9) M-1 and capacity 3700 fmol/ml serum. The presence in rabbit serum of a high affinity, GH-specific, binding protein raises important questions regarding its identity and possible physiological role in modulating the delivery and/or activity of GH in vivo.