The D2-like dopamine (DA) receptor family has continued to expand and now includes the D2-short (Das) and Dz-long (D2,,) receptor isoforms and the D3 and D4 receptors. The D2 receptor isoforms differ in length by 29 amino acids within the third cytoplasmic loop, a region of the receptor believed to be important for G protein coupling. This observation has led to the hypothesis that the two isoforms of the Dz receptor may utilize different signal transduction pathways when present in the same cell. The D2 and D3 receptors, although mostly different, show some common amino acid sequences within the third cytoplasmic loop. Thus, it is possible that the Dz and D3 receptors may employ similar signal transduction pathways. To test these hypotheses directly, NG108-15 neuroblastoma-glioma hybrid cells were stably transfected to express either the Dls, Dai,, or D3 DA receptors. All transfected but not untransfected NG108-15 cells demonstrated a dose-dependent reduction in the peak whole-cell potassium (K') current in response to receptor activation by DA or the DA receptor agonists quinpirole (QUIN) and apomorphine (APO). The modulation of K' current by Dzs receptor stimulation was prevented by pretreatment of the cells with cholera toxin (20 pg/ml for 18 h), whereas pertussis toxin pretreatment (500 ng/ml for 4 h) completely blocked the effects of DaL and D3 receptor activation. These observations suggest that the signal transduction mechanisms involved in coupling the two isoforms of the D2 receptor to the K' current are different, whereas the D2L and D3 receptor coupling mechanisms may be similar. In direct support of this hypothesis, it was observed that the intracellular application of a polyclonal antibody that is specific for the Gocy subunit completely blocked the ability of D2L and D3 receptors to modulate outward K* currents. In contrast, the DZs-mediated modulation of K' currents was blocked by intracellular application of a n antibody recognizing Gsct but not Goo. These findings demonstrate that Dzs and D2L receptors are able to couple to a common effector in a cell via two G protein pathways.