Objective: The aim of the present in vitro study was to examine the caries-inhibiting effect of a pit and fissure sealant (PFS) containing ion-releasing microcapsules under cariogenic conditions in a biofilm artificial mouth. Methods and Materials: Forty-eight human third molars were divided into four groups (n=12 per group). Fissures were extended with burs and sealed with experimental PFS. The four groups of specimens were treated as follows: 1) EPFS 1: EPFS (Premier Dental) of increasing viscosity, containing microcapsules loaded with remineralizing agents (calcium, phosphate, and fluoride ions); 2) US: fluoride-releasing PFS (UltraSeal XT plus, UltraDent Products, South Jordan, UT, USA); 3) EPFS 2: experimental PFS of constant viscosity containing microcapsules loaded with calcium, phosphate, and fluoride ions; and 4) FT: glass ionomer cement (GIC) (GC Fuji Triage CAPSULE WHITE glass ionomer cement, GC Europe NV, Leuven, Belgium). FT and US were used as control groups. EPFS 1 and EPFS 2 were the experimental groups. Specimens were stored in distilled water for 14 days at 37 degrees C, subjected to 10,000 thermocycles (5 degrees C and 55 degrees C) and finally exposed to microbiological cycling in a Streptococcus mutans-based artificial mouth for 10 days. Replicas were made for scanning electron microscopic (SEM) evaluation and specimens were cut for fluorescence microscopy. Results: Overall demineralization depths at the margin of Fuji Triage were significantly shallower than in the other groups (p<0.05). Overall demineralization depths adjacent to the experimental pit and fissure sealant EPFS 2 (59 +/- 15 mu m) were comparable to the values of the resin-based pit and fissure sealant UltraSeal XT plus (58 +/- 10 mu m, p >= 0.05). SEM revealed surface roughness of the GIC-based PFS. Conclusions: The experimental PFS with microcapsules containing active components for remineralization did not show a caries-inhibiting effect compared to a fluoride-releasing resin-based PFS. Lower demineralization depths adjacent to GIC sealants indicate an anticariogenic effect through fluoride ion release.