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AIM: To investigate the tolerance development against the relaxant effect of nitric oxide donating drug isosorbide dinitrate (ISDN) and sodium nitropruside(SNP) in internal anal sphincter (IAS) smooth muscle.METHODS: Relaxation responses of ISDN, and electrical field stimulation (EFS) were obtained before and after tolerance induction by ISDN incubation.RESULTS: ISDN (10-7-10-4 mol/L) and SNP (10-8-10-4mol/L) caused a concentration-dependent relaxation on the basal tonus of the isolated rabbit IAS strips. After a period of 2 h incubation of the 6 × 10-4 mol/L ISDN the relaxation effects of ISDN and SNP did not change compared to control strips. EFS evoked frequency-dependent relaxation in internal anal sphincter smooth muscle and Emax obtained from control strips were not changed in ISDN tolerance-inducing condition. In thisstudy nitrate tolerance was not observed in rabbit IAS smooth muscle.CONCLUSION: This result shows that nitric oxide donating drugs relaxes the internal anal sphincter of the rabbits without the development of tolerance.