Recombinant human dopamine D4.4 receptor-mediated G protein activation was characterized in membranes of transfected mammalian (Chinese hamster ovary) cells by the use of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding. An initial series of experiments defined the conditions (3 microM GDP, 100 mM NaCl, 3 mM MgCl2) under which optimal stimulation (2.2-fold increase in specific [35S]GTPgammaS binding) was achieved with the endogenous agonist dopamine. The number of dopamine-activated G proteins in Chinese hamster ovary-D4.4 membranes was determined through [35S]GTPgammaS isotopic dilution saturation binding, yielding a Bmax value of 2.29 pmol/mg. This compared with a D4.4 receptor Bmax value of 1.40 pmol/mg determined by [3H]spiperone saturation binding, indicating that 1 or 2 G proteins were activated per D4.4 receptor and that there were few or no "spare receptors" in this cell line. Under these conditions, the efficacy for stimulation of [35S]GTPgammaS binding at D4.4 receptors of 12 dopaminergic agonists was determined. Several antiparkinsonian drugs, including ropinirole, quinerolane and lisuride, exhibited agonist activity at D4.4 receptors (Emax = 74.3%, 72.4% and 32.2%, respectively, compared with dopamine = 100%). The EC50 values for agonist stimulation of [35S]GTPgammaS binding correlated well with the inhibition constants derived from competition binding with [3H]spiperone (r = +.99). However, other antiparkinsonian drugs (bromocriptine, L-DOPA and terguride) showed low affinity and/or were devoid of agonist activity at D4.4 receptors. The potency at D4.4 receptors of the novel, selective D4.4 receptor antagonist L 745,870 was determined, indicating that it has high affinity (Ki = 1.99 nM) without detectable agonist activity. Furthermore, L 745,870 completely inhibited dopamine-stimulated [35S]GTPgammaS binding with a Kb value of 1.07 nM. The action of an additional 20 chemically diverse dopaminergic ligands, including clozapine, ziprasidone, sertindole, olanzapine and several other "atypical" antipsychotics, in advanced development was investigated. Each of these ligands shifted the dopamine stimulation curve to the right in a parallel manner consistent with competitive antagonism at this site and yielding Kb values (32.6, 22.4, 17.2 and 26.5 nM, respectively) that agreed closely with their Ki values (38.0, 14.9, 18.5 and 26.1 nM). In contrast, raclopride and seroquel exhibited low affinity at D4.4 receptors (Ki > 1000 nM). Other compounds that showed antagonist activity at D4.4 receptors included the 5-hydroxytryptamine2A receptor antagonist fananserin (RP 62203), the sigma ligand BMY 14,802 and the D3 receptor antagonist GR 103,691. In conclusion, dopamine D4.4 receptor activity is unlikely to be an important factor in the clinical effectiveness of antiparkinsonian drugs, although low agonist efficacy at D4.4 receptors might be associated with a lesser incidence of side effects. Furthermore, antagonist activity at D4.4 receptors is a common property of many typical and atypical antipsychotic agents.