The effect of sphingosine-1-phosphate (S1P) on large conductance Ca²⁺-activated K⁺ (BK_Ca) channels was examined in primary cultured human umbilical vein endothelial cells (HUVECs) by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i), whole-cell membrane currents and single channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca²⁺]_i. [Ca²⁺]_i and membrane potentials were strongly correlated. In whole cell clamped cells, BK_Ca currents were activated by increasing [Ca²⁺]_i via cell dialysis with pipette solution and the activated BK_Ca currents were further enhanced by S1P. When [Ca²⁺]_i was buffered at 1 µM, the S1P concentration required to evoke half maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK_Ca channel activity in a reversible manner and shifted the relationship between Ca²⁺ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches, loading cells with GDP--S (1 mM) using a patch pipette, GDP--S application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK_Ca current and channel activation. These results suggest that S1P enhances BK_Ca channel activity by increasing Ca²⁺ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca²⁺ influx through Ca²⁺ entry channels. Inasmuch as S1P activates BK_Ca channels via a mechanism independent of G-protein coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca²⁺ mobilization in human endothelial cells.