Prolactin (PRL) and growth hormone (GH) have two distinct binding sites (Site 1 with high affinity; Site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of Site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands (hPRL and hGH) and their antagonists (hPRL-G129R and hGH-G120R) to (1) bind to PRLRs; (2) induce conformational changes in PRLRs; (3) activate signaling pathways associated with the PRLR; and (4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated JAK2/STAT5, Ras/Raf/MEK/Erk, and PI3-K/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be Site 1 plus Site 2 or two Site 1s, to elicit receptor mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GHR) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multi-functional therapeutics that target this class of cytokine receptors.