The mammalian prostate is densely innervated by hypogastric and pelvic nerves that play an important role in regulating the growth and function of the gland. While there has been much interest in the role of the noradrenergic innervation and adrenoceptors in prostate function, the role of cholinergic neurones in prostate physiology and pathophysiology is not well understood. This review focuses on the role of acetylcholine and cholinoceptors in prostate function. Nitric oxide, vasoactive intestinal polypeptide, and/or neuropeptide Y are co-localised with cholinesterase and/or acetylcholine transporter in some of the nerve fibres supplying the prostate. Their roles are also briefly discussed in this review. A dense network of cholinesterase-staining fibres supplies both prostate epithelium and stroma, suggesting a role of acetylcholine and/or co-localised neuropeptides in the modulation of prostatic secretions, as well as smooth muscle tone. A predominantly epithelial location for prostate muscarinic receptors indicated a major secretomotor role for acetylcholine. The muscarinic receptor subtype mediating muscarinic agonist-induced smooth muscle contraction or enhancement of contractions evoked by nerve stimulation differs in different species. In the human, there is evidence for M(1) receptors on the epithelium, M(2) receptors on the stroma, and both M(1) and M(3) receptors in some prostate cancer cell lines. Several recent investigations indicate that muscarinic receptors may also mediate or modulate normal, benign, and malignant prostate growth. The role of muscarinic agonists and their receptors and the influences of age, testicular, and other steroids in regulating the effects are reviewed.