The human D(2short) (D(2S)) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [3H]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [35S]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [35S]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D(2S) receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([3H]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [3H]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [3H]NPA binding with greater potency ( approximately 6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D(2S)/Go preparation. [3H]Spiperone binding occurred with an increased B(max) in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D(2S) receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein.