Substitution of arginine for glycine at position 120 in native 22-kDa human growth hormone (hGH) results in an analogue, G120R, which is unable to dimerize the GH receptor and is widely used to probe the molecular mechanism of action of hGH. When acting on human GH receptors, G120R antagonizes several biological effects of hGH, but is itself inactive as an agonist. It has been reported that this mutant also antagonizes hGH activation of the rat or human prolactin (PRL) receptor in cell-based assays, with no agonist activity. We have now tested this mutant in a sensitive MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-ESTA (eluted stain assay) bioassay using rat PRL receptors in the Nb2 cell line. We confirm that G120R acts as an efficient antagonist of native hGH, but show that it can also act as an agonist to generate intracellular signals leading to metabolic activation and proliferation of Nb2 cells. We have demonstrated an unusual sensitivity to the presence of zinc (Zn2+). In the absence of added Zn2+, G120R shows weak but full agonist activity in the bioassay, and this can be blocked by co-incubation with recombinant hGH-binding protein. G120R can therefore be utilized to discriminate between the molecular mechanisms of hGH interactions with its somatogenic and lactogenic receptors. Future studies with G120R in the rat may need to take account of its significant agonist effects on PRL receptors.