The large-conductance Ca(2+)-activated K(+) (BK) channel is activated by both the increase of intracellular Ca(2+) concentration and membrane depolarization. The BK channel plays crucial roles as a key molecule in the negative feedback mechanism regulating membrane excitability and cellular Ca(2+) in various cell types. Here, we report that a widely used slow-response voltage-sensitive fluorescent dye, bis(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(4)(3)], is a potent BK channel activator. The application of DiBAC(4)(3) at concentrations of 10 nM and higher significantly increased whole-cell BK channel currents in human embryonic kidney 293 cells expressing rat BK channel alpha and beta1 subunits (rBKalphabeta1). In the presence of 300 nM DiBAC(4)(3), the activation voltage of the BK channel current shifted to the negative direction by approximately 30 mV, but the single-channel conductance was not affected. DiBAC(4)(3) activated whole-cell rBKalphabeta1 and rBKalphabeta4 currents in the same concentration range but partially blocked rBKalphabeta2 currents. The BK channel alpha subunit alone and some other types of K(+) channels examined were not markedly affected by 1 microM DiBAC(4)(3). Structure-activity relationship analyses revealed that a set of oxo- and oxoanion-moieties in two 1,3-dialkylbarbituric acids, which are conjugated by oligomethine, is the novel skeleton for the beta-subunit-selective BK channel-opening property of DiBAC(4)(3) and related oxonol compounds. This conjugated structure may be located stereochemically in one plane. These findings provide a molecular and structural basis for understanding the regulatory mechanism of BK channel activity by an auxiliary beta subunit and will be fundamental to the development of beta-selective BK channel openers.