The effect of the antifungal drug miconazole on Ca^(2+) signaling in human breast cancer cells is unknown. This study examined the effect of miconazole on cytosolic free Ca^(2+) concentrations ([Ca^(2+)]i) in ZR-75-1 human breast cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca^(2+)]i. Miconazole induced [Ca^(2+)]i rises concentration-dependently. The response was reduced by 60% by removing extracellular Ca^(2+). Miconazole-induced Ca^(2+) entry was abolished by the protein kinase C (PKC) inhibitor GF109203X, and nifedipine, but was insensitive to econazole, SKF96365 and the protein kinase C activator phorbol 12-myristate 13 acetate (PMA). In Ca^(2+)-free medium, treatment with the endoplasmic reticulum Ca^(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin (TG) greatly inhibited miconazole-evoked [Ca^(2+)]i rises. Conversely, treatment with miconazole abolished TG and BHQ-evoked [Ca^(2+)]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished miconazole-induced [Ca^(2+)]i rises. At concentrations of 30-50 μM, micronazole killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca^(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxy methyl (BAPTA/AM). Together, in ZR-75-1 cells, miconazole induced [Ca^(2+)]i rises by evoking PLC-dependent Ca^(2+) release from the endoplasmic reticulum, and PKC-regulated nifedipine-sensitive Ca^(2+) entry. Miconazole-caused cell death was not triggered by a preceding [Ca^(2+)]i rise.