An increase in fluorescence is observed upon light activation of bovine rhodopsin. The rate of increase is monoexponential (t1/2 = 15.5 min) at 20 degrees C in 0.1% lauryl maltoside, pH 6.0, and parallels the rate of decay of metarhodopsin II. We show that the increase in fluorescence is due to the release of free retinal, which no longer quenches the tryptophan fluorescence. An extrinsic fluorescence reporter group, pyrene maleimide, attached to bovine rhodopsin also shows an increase in pyrene fluorescence on illumination. The rate of increase in pyrene fluorescence matches the rate of increase in tryptophan fluorescence. This result has been used to develop a micromethod, requiring on the order of 1 microgram of rhodopsin, for measurement of metarhodopsin II decay. An Arrhenius plot derived from the fluorescence assay shows the energy of activation barrier for retinal release from rhodopsin to be 20.2 kcal/mol in 0.1% dodecyl maltoside at pH 6.0.