Cytokines that interact with receptors of the hematopoietin super-family have recently been reported to stimulate receptor-associated JAK tyrosine kinases, including PRL activation of JAK2. Unlike other tyrosine kinases, none of the JAK kinases has thus far been implicated in oncogenesis, and their involvement in growth signaling has not been established. Using the PRL-dependent pre-T-cell line Nb2, the present study provided a link between bivalent dimerization of a hematopoietin receptor and activation of its associated JAK kinase, and demonstrated a strong positive correlation between the mitogenic potency of a series of bivalent anti-PRL receptor antibodies and the degree of induced tyrosine phosphorylation of JAK2. Antibody bivalency was required for JAK2 phosphorylation. Monovalent anti-PRL receptor Fab fragments alone were inactive, but their activity could be partially restored by cross-linking with bivalent anti-Fab antibodies. Additional evidence for antibody-induced receptor dimerization was provided by a bell-shaped dose-response curve for the most potent receptor agonist, monoclonal antibody T6. This phenomenon is typically seen at pharmacological concentrations of bivalent ligands, when bound ligand molecules fail to adjoin a second receptor due to occupancy. The present study provided functional support for a model of PRL receptor triggering by ligand-induced receptor homodimerization and subsequent activation of the associated tyrosine kinase JAK2.