Circulating GH consists of several molecular size species with different biological activity. A reduced sensitivity of some monoclonal antibodies towards high-molecular weight GH variants has been reported. The aim of the present work was to evaluate the molecular size species of circulating GH using Sephadex G-100 gel filtration chromatography in acromegalic patients and in normal subjects employing both RIA and an immunoradiometric assay for all GH determinations. In 6 normal subjects, studied under GHRH stimulation, little GH was 69.8±6% (mean ±sd), big GH (44 kD) 26.4±6% and big-big GH (>80 kD) 2.8±4%, in IRMA, with a good correspondence with RIA results (70.8±8, 27.0±4, and 3.2±2%, respectively). In 13 untreated acromegalic patients, studied in basal conditions, the little form constituted 76.2±7%, the big form 18.3±4%, which is significantly lower than in normals (p<0.05), and the big-big form 5.5±7%. Similar results were obtained with RIA. A clear elevation of big-big GH (21% for both in IRMA, and 15.7 and 27.8% in RIA) was found in 2 patients with IGF-I levels lower than expected on the basis of mean GH concentrations. The study was extended to an additional acromegalic patient, previously operated and irradiated on, characterized by discrepant serum GH levels in RIA (4.6 μg/l), and in IRMA (1.4 μg/l), and by normal IGF-I levels. Serum GH showed a lack of parallelism to standard GH in RIA, but not in IRMA. RIA immunoreactivity was almost completely composed (92%) of a high molecular weight GH form (>90 kD), not recognized by IRMA. All IRMA immunoreactivity eluted with a Kav corresponding to 19–50 kD. In conclusion: a. the three main molecular size isomers of serum GH are similarly recognized by IRMA and RIA methods in normal subjects. b. in acromegaly, both quantitative and qualitative modifications of the GH chromatographic profile may be present. In particular, increased amounts of big-big forms, whether or not recognized by monoclonal antibodies, have been observed. Their lower bioactivity, suggested by the normal or lower than expected IGF-I levels, can account for the discrepancy between serum GH levels and the clinical picture or IGF-I levels sometimes observed in acromegaly.