The membrane potential change triggered by photoisomerization of various spiropyrans was investigated. All spiropyrans, except for the spiropyran with a fluoric substituent (SPF), induced a membrane potential change in the presence of an electrolyte concentration gradient, while only the spiropyrans with ester groups induced a membrane potential change in the absence of an electrolyte concentration gradient. The result arose from the difference in the sites where the spiropyrans were located in the membranes. The photoinduced membrane potential change was also dependent on the physical properties of membranes such as hydrophilicity. Both the location sites of the spiropyrans and the mobility of the electrolyte ions are important factors determining the photoinduced membrane potential change.