With the use of fura 2 measurements in multiple and single cells, we examined whether cysteinyl leukotrienes (CysLT) mediate angiotensin II (ANG II)-evoked increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in neonatal rat cardiomyocytes. ANG II-evoked CysLT release peaked at 1 min. The angiotensin type 1 (AT₁) antagonist losartan, but not the AT₂ antagonist PD-123319, attenuated the elevations in [Ca²⁺]_i and CysLT levels evoked by ANG II. Vasopressin and endothelin-1 increased [Ca²⁺]_i but not CysLT levels. The 5-lipoxygenase (5-LO) inhibitor AA-861 and the CysLT₁-selective antagonist MK-571 reduced the maximal [Ca²⁺]_i responses to ANG II but not to vasopressin and endothelin-1. While MK-571 reduced the responses to leukotriene D₄ (LTD₄), the dual CysLT antagonist BAY-u9773 completely blocked the [Ca²⁺]_i elevation to both LTD₄ and LTC₄. These data confirm that ANG II-evoked increases, but not vasopressin- and endothelin-1-evoked increases, in [Ca²⁺]_i involve generation of the 5-lipoxygenase metabolite CysLT. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] antagonist 2-aminoethoxydiphenyl borate attenuated the [Ca²⁺]_i responses to ANG II and LTD₄. Thus AT₁ receptor activation by ANG II is linked to CysLT-mediated Ca²⁺ release from Ins(1,4,5)P₃-sensitive intracellular stores to augment direct ANG II-evoked Ca²⁺ mobilization in rat cardiomyocytes.