Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constrants estimated for TMR-labeled GTF at pH 6.8 and 25°C in the absence and presence of sucrose were 9.2 and 13.3 s¹, respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (25 s¹) under comparable conditions.