The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5-9.0 mug of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95degreesC) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85degreesC, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate). The stability of GFPuv in Tris-HCl (pH > 8.0) was constant at 90 and 95degreesC, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100degreesC.