The thermally induced aggregation of bovine beta-lactoglobulin in the glass state was studied both as the pure protein and in trehalose mixtures. Calorimetric measurements and size exclusion chromatography were used to characterize the glass state and aggregation, respectively. The initial aggregation rate was characterized by the initial rate of tetramer formation (beta-lactoglobulin dimerizes under the elution conditions), which showed Arrhenius temperature dependence with an activation energy of 95 kJ mol(-1) in the temperature range 60-100 degrees C. The trehalose addition slowed the aggregation in a way that depended exponentially on volume fraction, exp(-phi/phi*). The volume fraction characterizing the retardation of the reaction, phi*, was 0.173 of the order required to fill the interstices between close-packed protein molecules. In this system, trehalose was acting as an inert glass-forming diluent. In a general way, this supports the water substitute hypothesis and the understanding of the mechanisms underlying anhydrobiosis and protein stabilization technologies.