1. We have studied the effects of the metabolic inhibitors cyanide (CN) and deoxyglucose (DG) on the intracellular Ca2+ concentration ([Ca2+]i) in macrovascular endothelial cells derived from human umbilical vein (EA cells). 2. CN- and DG increased [Ca2-]i in non-voltage clamped cells. This effect was dependent on extracellular Ca2+ concentration and membrane potential, indicating that CN- induced a Ca2+ entry. 3. During expose to CN- and/or DG, EA cells depolarise. This depolarisation is sometimes preceded by a small, but transient hyperpolarisation due to activation of a big - conductance K+ channels, BKCa, present in EA cells. However, in approximately 90% of the cells tested, the CN- and/or DG induced elevation of [Ca2+]i was insufficient to activate BKCa. 4. CN- and/or DG enhanced BKCa currents preactivated by an elevation of [Ca2+]i via cell dialysis with 0.5 and 1 microM, respectively. Thus, metabolic inhibition sensitises BKCa. 5. The CN- induced depolarisation of EA cells occurs by activating a current that reversed at positive membrane potentials. Substituting extracellular cations abolished the inward component of this current by NMDG, indicating that CN- activated a non-selective cation channel, NSC. This current was reduced by extracellular Ca2+ and Mg2'+ but is partially carried by Ca2+. 6. It is concluded that CN elevates [Ca2+]i by activating Ca2+ permeable NSC channels. The properties of these channels are similar to those of the recently described trp3 channels expressed in endothelium.