The patch-clamp technique was used to examine the inhibition of delayed rectifier K⁺ channels by agents that release intracellular Ca²⁺. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 μmol/L) present to inhibit delayed rectifier K⁺ current (I_K(dr)) and Ca²⁺-activated Cl⁻ current (I_Cl(Ca)), angiotensin II (Ang II) and caffeine increased Ca²⁺-activated K⁺ current (I_K(Ca)) between −25 and 80 mV (n=5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 μmol/L) present to inhibit I_K(Ca) and I_Cl(Ca), Ang II and caffeine only caused inhibition of I_K(dr). Block was achieved within 15 seconds of drug application and was reversible upon washout (n=5). The effects of Ang II on I_K(Ca) and I_K(dr) were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n=3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both I_K(Ca) and I_K(dr). In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 μmol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26±2.9 mV depolarization from −54±3.1 to −28±1.7 mV (n=6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca²⁺ indicator indo 1 (100 μmol/L), Ang II, caffeine, and 4-AP increased [Ca²⁺]_i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca²⁺]_i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca²⁺]_i (n=4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca²⁺-activated K⁺ channels, a 45±7-picosiemen 4-AP–sensitive K⁺ channel was identified that was sensitive to cytoplasmic Ca²⁺ (n=6). Increasing intracellular Ca²⁺ decreased channel opening probability [N×P(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca²⁺ from <5 to 200 and 600 nmol/L free Ca²⁺ decreased N×P(open) by 52±3% and 73±7%, respectively (n=6). The decrease in opening probability of the delayed rectifier K⁺ channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively. These results suggest that agonist-induced changes in intracellular Ca²⁺ can alter vascular smooth muscle membrane potential through regulation of delayed rectifier K⁺ channels.