The purpose of the study was to assess the usefulness of environmental scanning electron microscopy (ESEM) to evaluate stain removal from extracted teeth. The ESEM differs from conventional SEM in that no sample preparation is needed, eliminating artifactual changes. Furthermore, the same sample can be viewed on multiple occasions, allowing "before" and "after" pictures of the same tooth. As a model stain removal device, we tested the Sonicare sonic toothbrush, which has previously been shown to remove dental stain in vivo. Twelve freshly extracted teeth with extrinsic coffee, tea or tobacco stain were obtained for the study. Nine of these had heavy stain (stain covering more than one-third buccal or lingual surface) and were used without further modification. Three teeth were treated in vitro with chlorhexidine and a mixture of coffee and tea to enhance staining. All teeth were examined by ESEM at three times: prior to brushing, after 15-30 seconds of brushing, and after 60-80 seconds of brushing. Light microscopy and 35 mm photography was also done to correlate the ultrastructural changes with those visible at low magnification. Water, mouthwash and 30% slurry of toothpaste were used as fluid vehicles during brushing, but little difference in stain removal was noted among these three fluids. Approximately half the stain was removed within 15-30 seconds, and most visible stain was removed in 60-80 seconds of brushing. Pits and crevices of tooth enamel that were smaller than the bristle diameter, and thus would be inaccessible to abrasive cleaning by direct bristle contact, were generally found to be stain-free. These findings confirm previous reports of the stain removal effectiveness of the Sonicare, and demonstrate the usefulness of ESEM for stain removal studies.