Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca²⁺ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 µM) increased intracellular calcium concentration ([Ca²⁺]_i) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca²⁺ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca²⁺]_i. These results suggest that ACh increases [Ca²⁺]_i in FSCs by activating phospholipase C, presumably through activation of M₁ receptors. The rise in [Ca²⁺]_i could trigger a variety of Ca²⁺-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.