1. The novel antimigraineur, dotarizine (30 microM), increased cytosolic Ca2+ concentration, [Ca2+]c, in fura-2-loaded bovine adrenal chromaffin cells. This increase was transient, reached a peak in about 2 - 5 min (0.53+/-0.07 microM; n=19) and then declined to basal levels over a further 5 min period. 2. This transient rise of [Ca2+]c was mimicked by 1 microM thapsigargin and by 30 microM cyclopiazonic acid (CPA), but not by 30 microM flunarizine. Both thapsigargin and CPA occluded the effects of dotarizine and vice versa. 3. All three compounds suppressed the transient [Ca2+]c rises induced by caffeine (10 mM, 10 s); blockade induced by thapsigargin was irreversible and that induced by CPA and dotarizine was reversible. 4. Of the three compounds, only dotarizine blocked reversibly the [Ca2+]c spikes induced by short pulses of high K+ (70 mM, 5 s), suggesting that dotarizine blocks voltage-dependent Ca2+ channels but CPA and thapsigargin do not. 5. Dotarizine caused a gradual and reversible depletion of endoplasmic reticulum (ER) Ca2+ in chromaffin cells transfected with ER-targeted aequorin. CPA had a similar effect. 6. These data show that dotarizine shares with thapsigargin and CPA the ability to deplete Ca2+ in the ER; this novel action of dotarizine could be relevant to its prophylactic effects in migraine. Unlike thapsigargin and CPA, however, dotarizine additionally and reversibly blocks Ca2+ entry through voltage-dependent Ca2+ channels.