Spectral properties of a recently developed voltage-sensitive dye, di-4-ANEPPDHQ, were characterized as the dye was dissolved in the solvent dimethyl sulfoxide as the stock solution, in Hank's buffered salt solution as the staining solution, and bound to the plasma membrane of primary rat hippocampal neurons and immortalized mouse hypothalamic neurons (GT1-7) in vitro. Their dependence on the local chemical and electrical environment of dye molecules was determined. The excitation and emission peaks are 479 nm and 570 nm for the stained primary neurons, and 476 nm and 585 nm for the stained immortalized neurons. The excitation and emission bands of the stained GT1-7 neurons, defined as 50% peak intensity, are 429-516 nm and 544-648 nm, respectively.