Recent advances in fluorescence microscopy techniques have allowed the video-time imaging of single molecules of fluorescent dyes covalently bound to proteins in aqueous environments1. However, the techniques have not been exploited fully because proteins can be difficult to label, and dye modification may cause partial or complete loss of activity. These difficulties could be circumvented by fusing proteins to green fluorescent protein (GFP) of the jellyfish Aequorea victoria. Here we report that single S65T mutant GFP molecules2 can be imaged using total internal reflection microscopy, and that ATP-driven movement of an individual kinesin molecule (a microtubule motor protein) fused to GFP can be readily observed.