1. Isolated rat neurohypophyses were stimulated electrically in an in vitro perifusion system. A Ca2(+)-sensitive microelectrode was placed in the centre of each neurohypophysis and [Ca2+]o decrease evoked by the stimulation were determined. 2. In neurohypophyses injected with Fura-2 AM (acetoxymethyl ester), increases and decreases in fluorescence excited at 340 and 380 nm, respectively, were evoked by stimulation. The time course of the fluorescence changes was similar to that of [Ca2+]o decreases, suggesting that the [Ca2+]o changes mirrored [Ca2+]i increases. 3. Calcium influx into neurosecretory axons and terminals was estimated as the difference in [Ca2+]o decrease rates immediately before and after train pulse stimulation. 4. Vasopressin release from the neurohypophysis, measured by specific radioimmunoassay, was facilitated by stimulation in parallel with a parameter of [Ca2+]o decrease multiplied by Ca2+ influx. 5. The O2 consumption rate, estimated as rate of PO2 decrease in the tissue, was facilitated by stimulation in parallel with [Ca2+]o decreases. 6. Possible calcium-dependent mechanisms of vasopressin release, and the energy-dependent step of the release by Ca2+, are discussed.