Aims: To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. Methods and Results: Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/- SEM) growth rate for 72-h biofilms (n = 4) was mu = 0.014 h(-1) (+/- 0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient > 0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. Conclusions: The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. Significance and Impact of the Study: The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.