– Five antiseptics commonly used in endodontic treatment were evaluated with regard to cytotoxicity and initial tissue irritating effect. The cytotoxicity was evaluated in a cell culture system using HeLa cells and the initial tissue irritating effect was assessed by application of the antiseptics onto an intact nonepithelialized tissue surface. The cell response was assessed by registering changes in the DNA synthesis of the cells and the tissue response was assessed by registering changes in the oxidoreductase enzyme activity of the tissue. To evaluate the ability of the cells and the tissue to overcome an induced damage, one part of the study included a recovery period before the condition of the cells and the tissue was assessed. The most severe cell and tissue reactions were caused by 5% Chloramine‐T® whereas no differences in toxicity or tissue irritating effect were found between 0.04% Jodopax®, 0.1% Bioscpt®, 0.1 % Hibitanc®, or 0.5% sodium hypochlorite. The HeLa cells showed the best recovery after having been influenced by Jodopax, Chloramine‐T or sodium hypochlorite solutions, whereas the best tissue recovery was observed in the experiments with Biosept and Hibitane.