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We used Fourier transform infrared spectromicroscopy in the attenuated total reflection configuration to study biochemical events associated with the response to light of an intact retina. We show that the technique is suitable for the detection in real time of molecular processes occurring in rod outer segments induced by light absorption. Two-dimensional correlation analysis was applied to the identification and interpretation of specific spectral changes associated to the evolution of the system. The technique allows us to observe an extensive protein translocation, which we interpret as arising from the release of transducin from the disk membrane and its redistribution from the outer segment towards the inner segment of rod cells. These results are in full agreement with our current understanding of retinal physiology and validate the technique as a useful tool for the study of complex molecular processes in intact tissue. [figure: see text] Spectral changes in the mid infrared region following exposure of an intact retina to light.