Acetylcholine (25 μmol/l) in the presence of the choline esterase inhibitor physostigmine (67 μmol/l) increased the release of growth hormone and efflux of 45Ca2+ from perifused bovine pituitary slices; the time taken for the maximal response to occur was the same. In batch incubations, acetylcholine (1 μmol/l–1 mmol/l) increased pituitary cyclic GMP concentrations in the pituitary gland within 2 min, and increased incorporation of [3H]inositol and [32P]phosphate into pituitary phosphatidyl inositol within 15 min. Cyclic AMP concentrations were not significantly changed 2 or 5 min after acetylcholine addition. All the tissue responses were inhibited by prior exposure of the tissue to atropine (1 μmol/l) but not by tubocurarine (10 μmol/l–1 mmol/l), indicating that the responses were mediated by receptors of the muscarinic type. The similarities between these responses and those to known hypothalamic hypophysiotrophic hormones are discussed.