How the endoplasmic reticulum (ER) and mitochondria communicate with each other and regulate plasmalemmal Ca²⁺ entry was studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ ([Ca²⁺]_i or [Mg²⁺]_i) and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in [Ca²⁺]_i produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺ and enhanced by raising extracellular pH. FCCP, dinitrophenol and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, whose first phase were accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin, but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺ free, EGTA solution and a Na⁺ free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was blocked partially by removing extracellular Ca²⁺ and fully by oligomycin, but not by thapsigargin or a Na⁺ deficient solution, accompanied by a rise in [Mg²⁺]_i, and summated with a high pH-induced rise in [Ca²⁺]_i, while the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺, but not by thapsigargin, depressed by decreasing Na⁺ and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺ free solution was depressed after FCCP actions. Thus, mitochondrial uncoupling causes Ca²⁺ release that activates Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, while Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes.