A yellow fluorescent protein (YFP) present in a strain of bioluminescent bacteria is shown here not only to modify the color and intensity of the emission, as already known and attributed to the interaction of YFP with a luciferase intermediate, but also remarkably to confer a negative temperature dependence to the in vitro system. The in vitro bioluminescence decay rate is actually independent of temperature in the range 5-25 degrees C, at approximately 1 microM YFP concentration. Several hypotheses are considered to explain this effect, based either on inactivation of YFP itself at higher temperatures or on its binding equilibrium with the luciferase intermediate. The first hypothesis is favored. Fluorescence anisotropy measurements show that YFP loses its chromophore at higher temperatures, but this alone cannot account for the negative temperature dependence. Gel chromatography shows the existence of an inactive YFP dimer, and the formation of more dimer at higher temperatures cannot be ruled out but is unlikely in our experimental conditions. Conformational changes may contribute to YFP inactivation. To our knowledge, there is no prior example of an enzymatic reaction in which the rate is slower at higher temperatures, within a physiological range.