The effect of trypsin on the photosynthetic electron transport of spinach chloroplasts has been investigated by measurements of the flash-induced absorption changes, indicating chlorophyll a1 at 703 nm, chlorophyll aII at 690 nm and at 515 nm via electrochromism the electrical potential gradient across the thylakoid membrane, respectively, and of the fluorescence induction caused by moderate actinic light. It was found: (1) In the presence of benzyl viologen as electron acceptor and with water as natural electron donor trypsin, incubation leads to a complete suppression of the absorption changes of the electrochromic effect and of chlorophyll aI and chlorophyll aII. (2) Addition of System I electron donors (N-methylphenazonium sulfate plus ascorbate or 2,6-dichlorophenolindphenol plus ascorbate) fully restores the chlorophyll aI photoreaction, whereas the initial amplitude of the electrochromic absorption change at 515 nm amounts about 50% of the control value without trypsin. The chlorophyll aII inhibition remains uneffected by System I electron donors. (3) System II electron donors (benzohydroquinone plus ascorbate or TPB) are unable to overcome the inhibition of electron transport by trypsin. (4) The fluorescence induction curve in 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea-blocked chloroplasts is modified by trypsin. The level of maximal fluorescence is remarkably decreased, whereas the initial fluorescence remains constant. The rise in kinetics is slightly decelerated. From these results, it is concluded that in the linear electron transport from water to benzyl viologen, mild trypsin treatment specifically attacks System II at a site very close to the reaction center, either on the oxidizing or on the reducing side. The reaction center of System II itself is relatively stable against trypsin. Arguments are presented which argue in favor of the trypsin attack being primarily directed at the reducing side of System II.