The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (μ) opioid receptor (MOR) and muscarinic acetylcholine receptor (mAChR) with Ca²⁺ channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole-cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M₂ mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC₅₀ values (nM) and maximal Ca²⁺ current inhibition (%) of 15.2 and 56.1, and 26.0% and 38.7%, respectively. Activation of MOR or M₂ mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca²⁺ currents via coupling with G_i/o protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M₂ mAChR, respectively. The prolonged stimulation of M₂ mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca²⁺ current inhibition, and was sensitive to the M₂ mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M₂ mAChR was not observed. These results suggest that in rat SPG neurons activation of M₂ mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow.