Seven highly purified dimeric forms of human pituitary growth hormone, composed of the monomeric forms 20 K hGH, 22 K hGH and 24 K hGH linked together by noncovalent or covalent bonds, have been characterized by an in vitro bioassay (the Nb2 assay), a radioreceptor assay and a radioimmunoassay. Considerable differences in the ability to displace labelled recombinant hGH were observed in the radioreceptor assay. The seven dimeric forms varied over a range between 22 K hGH (most effective) and 20 K hGH. The three covalently-linked dimeric forms had nearly identical affinity constants. The mitogenic action of all but one of the hGH dimers in the Nb2 assay was in the same mutual order as the receptor binding activity in the radioreceptor assay. In the RIA, all dose-response curves were parallel except for those obtained with 20 K hGH and with the dimeric form (20 K-20 K)hGH. In this assay, dimeric variants of the constituents 22 K hGH and 24 K hGH were approximately twice as active as 22 K hGH on a molar basis, suggesting about the same affinity between the antibodies and each of the monomeric forms. Determination of the amino acid compositions of the dimeric forms provided support for their content of monomeric constituents as established earlier by electrophoretic analysis.