Secondary caries is a major reason for the replacement of restorations. Because it is hypothesized that the development of secondary caries is closely associated with pathogenic oral bacteria, an in vitro microbial model has been developed to produce secondary carious lesions. A mixture of overnight cultures of Streptococcus mutans and Lactobacillus casei in dextrose-free trypticase soy broth, supplemented with 5% sucrose (TSBS), at 37 degrees C was used in this model as the inoculum for the experimental groups. Uninoculated control groups were incubated with medium only. Groups of human tooth specimens restored using composite, together with their respective controls, were exposed for 7 or 12 days to circulating cycles of TSBS (30 min each, 3 times per day) and a mineral wash solution (for a total of 22.5 h per day), at 37 degrees C. The pH of the experimental groups dropped to 4.l-4.5 during the test periods. The pH of the control groups remained at 6.8-7.0. The inoculated bacteria remained viable throughout the study. No contamination of experimental or control samples occurred. Laser scanning confocal microscopy demonstrated the development of incipient surface and wall lesions in all the specimens of experimental groups in as few as 7 days. Reproducibility of the model was confirmed in a second investigation. Therefore, it was concluded that this model can be used for studying the microbial etiology and prevention of secondary caries.