Binding putative muscarinic ([3H]-NMS and [3H]-QNB) or nicotinic ([3H]-cytisine) acetylcholine receptors was quantitatively studied through the use of in vitro binding experiments on either membrane preparations or brain sections of juvenile (3 months), mature (15 months) or senescent (23 months) cuttlefish. No specific binding could be detected with muscarinic receptor ligands under any of the experimental conditions employed (ligand concentrations, buffers, ionic charges, types of tissue, i.e., brain sections or membrane preparations). On the other hand, [3H]-cytisine demonstrated a specific and saturable binding with a single class of high affinity binding sites (Kd of 2.6–34.6 nM; Bmax of 128–1682 fmol/mg tissue equivalent, depending on the central structure). This binding was found to be heterogeneous throughout the central regions (optic lobe > pedal lobe; superior frontal lobe > … precommissural lobe; vertical lobe > … anterior basal lobe; subvertical lobe; inferior frontal lobe; median basal lobe). These results question the existence of muscarinic-like receptors in the cuttlefish brain, or at least of a pharmacological dissimilarity from vertebrate muscarinic receptors. In contrast, nicotinic-like receptors are widely present; interestingly, their density was found to be significantly reduced in most nervous central lobes of senescent cuttlefish when compared with mature animals. The most significant decrease (−71%) was found in the anterior part of the superior frontal lobe, which is involved in visual learning; this might be related to the changes, previously demonstrated, in cholinergic neurons in this lobe in the course of aging.