Evidence is emerging that oestrogen, besides acting via classical nuclear receptors, can rapidly influence the physiology of nerve cells through other mechanisms. Oestrogens have been shown to modulate the differentiation and function of embryonic midbrain dopaminergic neurones by stimulating neurite outgrowth, expression of tyrosine hydroxylase mRNA, dopamine uptake and release in spite of the fact that dopaminergic cells in the prenatal midbrain do not express the classical oestrogen receptor. This study therefore intended to unravel possible signal transduction pathways activated by oestrogen which might be associated with the above oestrogen effects. As a physiological second-messenger mechanism, we studied the influence of oestrogen on fluctuations of intracellular Ca2+ levels [Ca2+]i by microspectrofluorimetry of the Ca2+-sensitive indicator Fura-2, in primary cultures from embryonic mouse midbrains. 17Beta-estradiol (10 nM-1 pM) but not 17alpha-estradiol increased [Ca2+]i within 1-3 s in a dose-dependent way. Removal of extracellular Ca2+ abrogated K+-stimulated Ca2+ rise but did not affect 17beta-estradiol stimulation. Pretreatment of cells with thapsigargin (1 microM, 10 min), an inhibitor of Ca2+-pumping ATPases in the endoplasmic reticulum, abolished the 17beta-estradiol effect but not the K+-stimulated [Ca2+]i rise. Oestrogen effects on [Ca2+]i were completely mimicked by using a membrane-impermeant oestrogen-BSA construct. In order to identify oestrogen-sensitive cells, some cultures were subsequently immunostained for microtubule-associated protein II, tyrosine hydroxylase, or GABA. All oestrogen-sensitive cells were immunocytochemically characterized as neurones, and about half of these responsive neurones was found to be dopaminergic or GABAergic. These results demonstrate that 17beta-estradiol is capable of rapidly modulating physiological parameters of developing midbrain neurones by directly interacting with specific membrane binding sites coupled to a signal transduction mechanism that causes a calcium release from intracellular Ca2+ stores. It is suggested that oestrogen effects on differentiation and function of midbrain dopaminergic neurones are mediated by intracellular Ca2+ signalling.