The initial photoinduced charge separation in bacteriorhodopsin is shown to occur in <5 ps. This result is obtained by measuring the photovoltage rise time in an oriented film of bacteriorhodopsin (BR). A dye laser syncronously pumped by an Argon ion cw mode locked laser is used to produce 3-ps light pulses which, after passing through a dye amplifier chain, photoexcite the BR sample. The photovoltage transient is detected by an ultra-fast Josephson junction digital sampling oscilloscope with liquid-helium-cooled input circuitry. © 1990, The Biophysical Society. All rights reserved.