Histamine regulates a variety of physiological processes including inflammation, gastric acid secretion, and neurotransmission. The cellular response to histamine is subject to dynamic control, and exaggerated histamine reactivity in response to cysteinyl leukotrienes and other stimuli is important in a variety of different pathological conditions. The molecular mechanisms controlling histamine responsiveness are still unresolved. In investigating histamine responses in embryonic stem (ES5) and F9 embryonic carcinoma cells, we encountered a novel mechanism controlling the cellular reaction to histamine. Unstimulated cells displayed neither [3H]pyrilamine binding nor histamine-induced increases in cytosolic Ca2+ levels. Pretreatment of these cells, however, with leukotriene D4, leukotriene E4, serotonin, or fetal calf serum induced an immediate and transient ability of these cells to respond to histamine with an increase in cytosolic Ca2+ levels. This effect could be inhibited by pertussis toxin and was mimicked by GTP analogues. Importantly, the latter compounds also provoked immediate high affinity [3H]pyrilamine binding. We conclude that in these cells histamine responsiveness is directly controlled by pertussis toxin-sensitive G protein-coupled receptors, whose activation enables the H1 receptor to bind its ligand. These findings define a novel mechanism for regulating histamine H1 receptor activity and provide for the first time molecular insight into the mechanism by which cysteinyl leukotrienes and other external stimuli can increase histamine responsiveness.