Oestrogen plays an essential role in regulating growth and differentiation in the human endometrium which undergoes dynamic morphological and functional changes during the menstrual cycle in preparation for implantation. In this tissue, it has been suggested that intracellular calcium could be a key signal in transducing early responses to steroid hormones. Here, we have investigated the rapid effects of 17β-oestradiol on [Ca²⁺]i in a human endometrial cell line (RL95-2). Using confocal imaging microscopy, we show that physiological concentrations of 17β-oestradiol trigger rapid and transient increases in [Ca²⁺]i. Our results demonstrate that 17β-oestradiol-induced [Ca²⁺]i variations are critically dependent on calcium influx via lanthanum-sensitive calcium channels. Moreover, the 17β-oestradiol-induced Ca²⁺ influx is significantly increased by the depletion of intracellular stores by thapsigargin and decreased by chelerythrine chloride, an inhibitor of protein kinase C. These data indicate a non-genomic action of 17β-oestradiol to stimulate capacitative Ca²⁺ entry through store-operated calcium channels via a PKC-sensitive pathway.