The complexation between the photoreceptor sensory rhodopsin I (SRI) and its signal transducer protein HtrI was examined by assessing titration of the Schiff base chromophore of SRI with sodium hydroxide and reactivity with hydroxylamine in the presence or absence of HtrI. The apparent pK(a) of the protonated Schiff base of SRI is 12.2 in the presence and 9.5 in the absence of HtrI. Direct titration of the Schiff base proton was confirmed by titrating an artificial SRI reconstituted with a 14-fluororetinal which reduces the intrinsic pK(a) of the protonated Schiff base of the HtrI-complexed pigment from > 12 to 9.0. The SRI chromophore exhibits high stability to hydroxylamine bleaching in the presence of HtrI; however, removal of HtrI accelerates the bleaching rate 2.4-fold. These results indicate that SRI is physically associated with HtrI in its unactivated (i.e., dark) state. In view of the previously identified association of the SRI signaling state (S-373) With HtrI, we conclude that SRI transduces the signal to HtrI through its altered interaction with the prebound transducer protein. The effect of the altered SRI/HtrI interaction on resetting the signaling state of SRI was also examined. At neutral pH the decay of S-373 is retarded by 20-fold when HtrI is absent. This effect was found to be due to a raised enthalpic barrier for the transition state during S-373 decay. The energy barrier for S-373 decay in this pigment can be lowered by providing extramembranous protons (lowering bulk pH). Therefore, light-induced alteration in SRI/HtrI interaction is important for reducing the energy barrier for S-373 decay presumably by providing or assisting a proton supply for retinal Schiff base reprotonation.