Malonaldehyde formed by lipid oxidation is regarded as a main crosslinker in the formation of natural age pigment. To elucidate the mechanism of pigment formation the reaction of malonaldehyde with biomembranes using fluorescence spectroscopy has been studied. Rat liver mitochondrial ghosts or bovine serum albumin were reacted with malonaldehyde. In both cases two main fluorescence changes were observed: protein fluorescence decreased to 50% of its initial value in about two hours; aminoiminopropene fluorescence reached a maximum at a much slower rate. The kinetics support a two-step reaction hypothesis. First, malonaldehyde reacts with protein quenching its fluorescence. Next fluorescent interprotein aminoiminopropene (AIP) crosslinks are formed. The fluorescence lifetime value of the induced AIP fluorophore was shown to be similar to the lifetime of naturally occurring age pigment previously reported for mitochondrial ghosts prepared from aged animals (5.4 ns +/- 0.3 and 5.9 ns +/- 0.6, respectively).