ABSTRACT-The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltagesensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where a or a plus b 1 subunits of large conductance Ca 2+-activated K + (BK) channels were expressed (HEKBKa and HEKBKab). 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 HEKBKab was 10 -20 mV more negative than that in native HEK. In HEKBKa, the membrane hyperpolarization induced by 10 m M Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/FK even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBKab by application of 10 m M acetylcholine via increase in intracellular Ca 2+ 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.