Corticotropin-releasing factor (CRF-41) immunoreactive neurones in the CNS are widely distributed; although the major concentration is in the parvocellular division of the paraventricular nucleus, there are also large clusters of CRF-containing neurones in the brainstem, limbic system and cerebral cortex. In this study, we sought to determine whether dispersed rat brain cells cultured in vitro are capable of secreting CRF immunoreactivity. In both telencephalic and diencephalic cultures CRF content of the cells increased from day 7 to 28. This effect was greatest in cells raised in media containing 10% fetal calf serum and 10% heat inactivated horse serum as compared with media containing 10% fetal calf serum and 10% NuSerum. Exposure of the cells to 56 mM K+ or to 10(-9) M acetylcholine caused a release of CRF which was calcium dependent. The effect of acetylcholine was antagonised by atropine (10(-6) M). The CRF immunoreactivity in both types of cell produced two peaks on gel chromatography; one of which eluted with the void volume and the other of which co-eluted with standard CRF-41. However, only the mature peptide was detected in media from cells stimulated with potassium. We conclude that cell cultures of the rat diencephalon and telencephalon produce immunoreactive CRF-41 and that immunoreactive CRF-41 secretion in both diencephalic and telencephalic cells is regulated by acetylcholine.