Voltage-sensitive dyes (VSDs) provide a spatially resolved optical read-out of electrical signals in excitable tissues. Several common fluorescent VSDs display electrochromic shifts of their emission spectra, making them suitable candidates for ratiometric measurements of transmembrane voltages. These advantages of VSDs are tempered by tissue-specific shifts to their fluorescence emission. In addition, the optimal electrochromic dye response occurs in wavelength bands distinct from the dye’s maximal resting emission. This “action spectrum” can undergo tissue-specific shifts as well. We have developed a technique for in situ measurements of the action spectra of VSDs in intact excitable tissues. Fluorescence emission spectra of VSDs during action-potential depolarization were obtained within a single sweep of a spectrophotometer equipped with a change-coupled device (CCD) array detector. To resolve the subtle electrochromic shifts in voltage-induced dye emission, fluorescence emission spectra measured right before and during field-induced action-potential depolarization were averaged over about 100 trials. Removing white-noise contributions from the spectrometer’s CCD detector/amplifier via low-pass filtering in Fourier space, the action spectra of all dyes could be readily determined.