Objective: The purpose of this study was to detect the relative contribution of Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia in the production of oral malodor. Method and materials: The volatile sulfur compounds produced by these bacteria in vitro were measured semiquantitatively by a portable sulfide monitor. Results: Samples from the tongue, tonsils, and pharynx showed a significantly higher production (550 ppb) oi volatile sulfur compounds during the first 6 hours after anaerobic incubation in broths (brain-heart infusion, Columbia, and Trypticase Soy) than after incubation in agar media (300 ppb) (P 0.001). After 24 hours, values in broths and agars leveled off at 350 ppb (P = 0.3) and remained constant during the next 6 days. Measurement of separate pure cultures showed that maximal volatile sulfur compound production was reached 6 hours after incubation (450 ppb for the 3 bacteria). Higher volatile sulfur compound values were measured in brain-heart infusion. When measurements of mixed cultures of the 3 pathogens were performed every 15 minutes, the maximal value was reached after only 30 minutes of incubation (nearly 500 ppb). Conclusion: The in vitro volatile sulfur compound production of oral samples is preferably measured in broths. Maximal sulfur production from mixed cultures is reached after 30 minutes of incubation. Samples should always be inoculated at the same dilution. Keywords: culture media, Halimeter, halitosis, microbiology, volatile sulfur compounds