We have used phosphorescence from the luminescent chromophores N-acetyltryptophanamide (NATA) and erythrosin B (Ery B) to probe the molecular dynamics of the glassy state and the glass-to-rubber transition in amorphous sucrose. These measurements exploit the known sensitivity of phosphorescence intensity to the rate and extent of molecular collisions with the local environment. Both probes exhibit intense, well-defined phosphorescence emission spectra in the glassy sugar matrix. Time-resolved phosphorescence intensity decays for each probe were multiexponential both above and below the glass transition temperature, indicating that the sucrose matrix is heterogeneous on the molecular level; local microviscosities around the NATA probe were estimated in the range from 10(4) to 5 x 10(7) Pa . s in the glassy state at 25 degrees C. Plots of phosphorescence Lifetime versus temperature for both probes are linear above and below the glass transition temperature of sucrose (62 degrees C), but exhibit a break in slope near approximately 50 degrees C characteristic of a dramatic change in the rate of molecular collision between the probe and the local environment. The similar sensitivity of two chemically different spectroscopic probes (the indole ring of NATA and the xanthene ring of Ery B) to molecular changes near the glass transition in this sugar indicates that phosphorescence may provide a generally applicable, site-specific probe of the glassy state and changes in it in biological materials.