Objective: The objective was to evaluate the influence of rubber dam isolation on the response of human pulps capped with calcium hydroxide and an adhesive system. Methods and materials: Direct pulp capping was performed in 40 caries-free human premolars scheduled for extraction as part of orthodontic treatment. The teeth were randomly divided into 8 groups (n = 5) according to the combination of treatment factors: capping agent (adhesive resin or calcium hydroxide), isolation (rubber dam or cotton pellets only), and period of evaluation (30 or 60 days). Class 2 cavities were prepared, and pulp exposures were created on the occlusal floor. After capping, all teeth were restored with Z-100 placed incrementally. After 30 or 60 days, patients were asked about their symptoms, teeth were extracted, and serial sections were evaluated. The data were subjected to a nonparametric test. Results: Overall, the histologic features showed that the pulp response was worse for groups capped with adhesive. For adhesive-capped groups, the pulp response varied from pulp necrosis to acute inflammatory cell infiltrate, and most of the pulps capped without rubber dam isolation showed severe inflammatory cell infiltrate involving the coronal pulp with chronic abscesses. For the calcium hydroxide groups, all specimens showed dentin bridge formation, regardless of the type of isolation used. Conclusion: Calcium hydroxide should be used as the material of choice for pulp capping with or without rubber dam isolation. The use of adhesive systems in vital pulp capping is contraindicated, especially if rubber dam isolation is not implemented. Keywords: adhesive system, biocompatibility, calcium hydroxide, cytotoxicity, primer, pulp therapy, resin monomer, rubber dam