In this Letter, we address nanoscale heterogeneity in polymer mobility. Single molecule fluorescence lifetime fluctuations are exploited as a probe for local segmental dynamics in polymer films. Wide distributions in polymer mobility are observed, which depend on film fabrication, treatment and amount of solvent contained. Upon solvent evaporation mobile regions disappear, while more immobile regions settle, effectively increasing the glass transition temperature. Besides insight in polymer nanoscale dynamics and heterogeneity, our results bear specific relevance to photodynamic studies on single chromophores, where routinely polymer matrices are used for immobilization. (C) 2003 Elsevier B.V. All rights reserved.