The layered structure of Langmuir-Blodgett (LB) films of poly(vinyl pentanal acetal) (PVPe) has been investigated by the interlayer energy transfer from energy donor to acceptor moieties labeled to the polymer chain. The spatial order of multilayers was irreversibly disordered and mixed by the thermal treatment. The thermal relaxation clearly occurred at temperatures higher than the glass transition temperature. Theoretical calculations based on Forster kinetics were applied to this phenomenon, which was successfully represented by assuming a Gaussian distribution of chromophores in the direction normal to the layer plane. The fitting with the experimental data allowed us to evaluate the diffusion constant of polymer segments on the order of 10(-16)-10(-18) cm2/s and the apparent activation energy of 150 kJ/mol. The energy-transfer method could probe the diffusion process of polymer segments in the ultrathin LB films.