Prolactin (PRL) acts through the long form (LF) of the human PRL receptor (hPRLR) to cause differentiation of mammary epithelial cells through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway and subsequent transcriptional events. To determine whether the inhibitory action of hPRLR short forms (SFs; S1a and S1b) on PRL-induced signal transduction through the LF results from heterodimerization, we studied complex formation among variant forms of the hPRLR. 3′-Tagged fusion constructs, with activities comparable to the wild-type species, were used to investigate homodimer and heterodimer formation. The LF and both SFs of the hPRLR formed homodimers under nonreducing conditions, independently of PRL, but formed only monomers under reducing conditions. Coimmunoprecipitation of the cotransfected LF with the SFs (S1a or S1b) in transfected cells showed ligand-independent heterodimerization of individual SFs with the LF. Bioluminescence resonance energy transfer analysis demonstrated homo- and heterodimeric associations of hPRLR variants in human embryonic kidney 293 cells. Biotin-avidin immunoprecipitation analysis revealed that hPRLR forms are cell surface receptors and that SFs do not influence the steady state or half-life of the LF. Significant homo- and heterodimerization of biotinylated membrane hPRLR forms was observed. These findings indicate that homo- and heterodimers of hPRLR are constitutively present, and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal transduction configuration. Although SF homodimers and their heterodimers with LF mediate JAK2 activation, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/signal transducer and activator of transcription 5 pathway. This prevents the heterodimeric LF from mediating activation of PRL-induced genes.