We investigated the effects of 1,25-dihydroxyvitamin D₃ [25(OH)₂D₃] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)₂D₃ and several of its analogs. The results showed that 1,25(OH)₂D₃ induced tPA secretion at 10¹⁰ to 10¹⁶ M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca²⁺ channels, Ro-24-5531, were ~10% as active as 1,25(OH)₂D₃. An analog that binds the vitamin D receptor poorly but is an effective Ca²⁺ channel agonist, Ro-24-2287, required ~10¹³ M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)₂D₃. Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca²⁺]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)₂D₃ was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)₂D₃ on two different induction pathways of tPA release.