Functional cell-free systems may be excellent tools with which to investigate light-dependent signal transduction mechanisms in plants. By evacuolation of parsley protoplasts and subsequent silicon oil gradient centrifugation of lysed evacuolated protoplasts, we obtained a highly pure and concentrated plasma membrane-containing cytosol. Using GT- and G-box DNA elements, we were able to demonstrate a specific localization of a pool of G-box binding activity and factors (GBFs) but not one of GT-box binding activity in this cytosolic fraction. The DNA binding activity of the cytosolic GBFs is modulated in vivo as well as in vitro by light and phosphorylation/dephosphorylation activities. The regulation of cytosolic G-box binding activity by irradiation with continuous white light and phosphorylation correlates with a light-modulated transport of GBFs to the nucleus. This was shown by a GBF-antibody cotranslocation assay in permeabilized, cell-free evacuolated parsley protoplasts. We propose that a light-regulated subcellular displacement of cytosolic GBFs to the nucleus may be an important step in the signal transduction pathway coupling photoreception to light-dependent gene expression.