Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor: hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.Human prolactin (hPRL), 2 growth hormone (hGH), and placental lactogen (hPL) are hormones belonging to the class 1 cytokine family (1). These three proteins share significant sequence homologies; the more closely related hPL and hPRL display 85% sequence identity, and hPRL shares a 23% identity with hGH. Each of these four-helix bundle proteins is recognized as a lactogenic hormone based on their ability to bind and activate primate prolactin receptors (2). The binding reaction that takes place between lactogenic hormones and the prolactin receptor results in the formation of a heterotrimeric complex with a 2:1 ratio of receptor to hormone (3) (Fig. 1). The formation of this ternary complex is believed to proceed in an ordered process in which the N-terminal S1 subdomain of the prolactin receptor first binds at a hormone surface comprised of elements of helices 1 and 4 as well as the sequence connecting helices 1 and 2 (site 1) before subsequently binding a second prolactin receptor by its S1 subdomain at a hormone surface composed of elements of helices 1 and 3 (site 2). During secondary receptor binding, an additional receptor-receptor binding surface is formed between the C-terminal S2 subdomains of the membrane-bound receptors and is distal to the bound hormone. Thus, for site 2 the ⌬G is divided between the hormone/ receptor interfaces and the two interacting S2 receptor surfaces. The formation of this extracellular trimolecular complex orients the intracellular domains of the...