The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC₄(3)), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where α or α plus β1 subunits of large conductance Ca²⁺-activated K⁺ (BK) channels were expressed (HEKBKα and HEKBKαβ). The fluorescent intensity of DiBAC₄(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC₄(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBKαβ was 10 - 20 mV more negative than that in native HEK. In HEKBKα, the membrane hyperpolarization induced by 10 μM Evans blue, a BK channel opener, was detected with DiBAC₄(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/F_K even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBKαβ by application of 10 μM acetylcholine via increase in intracellular Ca²⁺ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC₄(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC₄(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.