The distribution of [3H]acetylcholine ([3H]ACh) and [3H]ACh co-incubated with 1-mM nicotine (muscarinic receptor), and [3H]ACh co-incubated with 1.5 microM atropine (nicotinic receptor) binding sites were studied in man and compared to monkey, cat and rat using quantitative in vitro autoradiography. The highest density of total [3H]ACh binding sites was found in laminae II-III, IX (motor neuron areas) and X close to the central canal. The distribution pattern of the muscarinic cholinergic binding sites was similar to that of the total cholinergic binding. In general the number of nicotinic binding sites in the spinal cord was relatively small. The largest number of such binding sites was found in laminae II-III of the dorsal horn and in laminae X around the central canal. It is evident that the spinal cord has a 2-3 times higher number of muscarinic than of nicotinic cholinergic receptors.