Oestrogen plays a key role in a great variety of actions in the nervous system, either through classical or alternative pathways. The classical pathways are initiated after oestrogen binding to the oestrogen receptors ER or ER, which translocate from the cytoplasm to the nucleus and act there as transcription factors. Alternative pathways are initiated at the plasma membrane and cytoplasm, via binding to classical or non-classical ERs. Using isolated ciliary ganglion neurons from the chick embryo and Ca²⁺ imaging, we demonstrated that a 10-min exposure to 17-oestradiol reduces Ca²⁺ influx through the plasma membrane. This effect was not reproduced by oestradiol conjugated to bovine serum albumin, which does not cross the plasma membrane, indicating that 17-oestradiol was acting intracellularly. ER was detected in the cytoplasm by immunostaining and its involvement in the regulation of Ca²⁺ influx by ICI182,780 inhibition. The phosphatidylinositol-3 kinase (PI3-kinase) inhibitor wortmannin and the nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (L-NAME) both blocked the oestradiol effect. The oestradiol effect was reproduced by 8Br-cGMP and abolished in the presence of the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Our study indicates that 17-oestradiol can regulate Ca²⁺ influx via PI3-kinase, NOS and PKG after activation of cytoplasmic ER.