Cholinergic and serotonergic neurons of the rostral pontine reticular formation have been implicated by animal studies in the modulation of sleep and waking. To define better the spatial relationships between muscarinic and serotonergic receptor binding in the rostral human brainstem, we used 3-dimensional computer reconstructions of serial autoradiographs generated with radioligands to muscarinic and serotonergic receptors. Receptor binding was assessed in a series of 9 human infants, and 3-dimensional reconstructions were performed in a representative infant at 53 postconceptional weeks. The computer reconstructions demonstrated a 3-dimensional distinct pattern in the rostral pontine reticular formation, with high (3H)lysergic acid diethylamide binding to serotonin receptors in the median raphe nucleus flanked by paramedian bands of high (3H)quinuclidinyl benzilate binding to muscarinic cholinergic receptors in the medial nucleus pontis oralis. Based upon comparisons to animal data, we suggest that the muscarinic-serotonergic pattern of receptor binding in the rostral pontine reticular formation represents part of the neurochemical organization of the circuitry involved in the modulation of rapid eye movement (REM) sleep in humans.