To examine whether GTP-binding proteins (G proteins) mediate the ability of neurotensin to lower the affinity of dopamine D2 agonist binding, the modulation by neurotensin in vitro of N-[3H]propylnorapomorphine [( 3H]-NPA) binding was investigated following pretreatment with pertussis toxin and N-ethylmaleimide in rat neostriatal membranes. Preincubation with N-ethylmaleimide (100 microM) markedly inhibited pertussis toxin-induced back-ADP ribosylation of three proteins with apparent molecular masses of 41, 40, and 39 kDa, respectively. This inhibition was prevented by adding dithiothreitol (250 microM) during the preincubation. N-Ethylmaleimide increased the KD (180 +/- 30%) and decreased the Bmax (-31 +/- 9%) of [3H]NPA binding sites but did not affect the binding properties of the selective D2 antagonist [3H]raclopride. N-Ethylmaleimide pretreatment did not affect the neurotensin (3 nM)-induced increase in the KD of [3H]NPA binding sites. Pertussin toxin treatment in vivo and in vitro was similarly ineffective. In conclusion, the present study indicates that neurotensin modulation of D2 agonist binding in neostriatal membranes is not mediated by G proteins.