Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment ( | Δ μ → | =19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis → trans isomerization of the pB cofactor and the regeneration of pG. The large | Δ μ → | , reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light.