The aim of this study was to investigate the morphological features and distribution of biofilms on Invisalign orthodontic appliances, in a sample of 'slow' and 'fast' plaque formers using scanning electron microscopy (SEM). Fifty-six Chinese male/female volunteers (aged 19-39 years) were screened for their plaque-forming rate using the plaque percentage index (PPI) coupled with digital photography and computer-based image analysis, after a period of 48 hours of abstinence from oral hygiene procedures. Eleven volunteers (seven males/four females) representing the lowest and highest ends of the plaque formation spectrum were chosen as slow and fast plaque formers, respectively. The subjects wore a full-coverage splint appliance, in which four tiles of Invisalign material were embedded. These tiles were collected at intervals of 1, 3, 6, 12, 24, and 48 hours, as well as 3, 7, and 14 days, immediately fixed in 10 per cent paraformaldehyde in 0.2 M cacodylate buffer solution and prepared for SEM. The surface configuration of the Invisalign appliance was visualized, as well as the chronological pattern of biofilm formation. Significance between fast and slow plaque formers was determined using a Student's t-test. Colonization appeared to centre initially on the raised edges or textured surfaces of the appliance, and initial adhesion was quicker and more abundant in the fast plaque-forming group. In the later stages of biofilm development, both groups showed no discernible differences in biofilm accrual on the surfaces, but the fast group displayed a more complex biofilm structure. More recessed and sheltered areas of the appliance, such as the cusp tips and attachment dimples, harboured more biofilm than the flat surfaces. Hence, it seems that the novel Invisialign orthodontic appliance is a useful tool to investigate the features of biofilm formation in time-course studies.