The stimulation of muscarinic receptors induces the activation of nonselective cation (NS) channels, in an internal ATP-dependent manner, in guinea-pig adrenal chromaffin cells. Therefore, we attempted to determine with fluorescent techniques in which part of the cell membrane muscarinic receptors (R) and NS channels were localized. The m4AChR was immunodetected in the adrenal medulla and not in the cortex. A three-dimensional analysis of dissociated chromaffin cells revealed that the cell comprised a nuclear portion that contained mainly the nucleus and a nonnuclear portion. The m4-like immunoreactivity (IR) was mainly distributed in the membrane area in the nuclear portion, and the majority of m4-like IR was closely associated with nicotinic AChR-like IR in double stainings. The intracellular concentration of Na+, as measured with SBFI dye, increased in response to muscarine, and this increase was larger in the nuclear portion than in the non-nuclear portion. We thus concluded that m4 receptors and probably NS channels are distributed in the plasma membrane near the nucleus.