Confocal microscopy and vital fluorescence techniques were combined for the first time to investigate ex vivo human dental plaque. The vital fluorescence technique used discriminates vital from dead cells, while confocal laser scanning microscopy allows the optical sectioning of undisturbed biofilms leaving the samples intact during analysis. The concomitant use of both methods made an examination of the three-dimensional architecture of dental plaque possible. The topography of plaque biofilms that were allowed to accumulate in situ on glass and enamel was recorded. The distribution of plaque microflora vitality as well as its accumulation varied according to plaque age. A plaque thickness of up to 8, 35 and 45 microm was estimated ex vivo on enamel after 1, 2 and 3 days, respectively. Young and sparse plaque biofilms consisted mainly of dead material. Vital bacteria were observed on top of this dead layers.