Introduction Nowadays, an increased demand for various surgical operations has raised a need of knowledge over principles and methods used to induce local and general anesthesia. Nevertheless, it is also necessary to gain a deep knowledge over positive or negative interactions of the endogenous biogenic substances on the process of general anesthesia. NO is one of these chemicals that has been paid a special attention during last 30 years. Materials and methods To study the role of NO in the process of general anesthesia and, meanwhile, gaining a suitable method in the administration of anesthetic agents as well as finding a new combination to induce anesthesia, three drugs were administered to mice prior to sodium thiopental injection. For this purpose, 60 male mice were used in three periods of studies (20 animals per study). Every 20 animals were divided into four experimental groups of five each. Each group received either of the following drugs via the subcutaneous route once daily for 3 days: l -arginine (a precursor of NO, 10 mg/kg), nitroglycerine (a donor of NO, 1 mg/kg), methylene blue (an inhibitor of guanylyl cyclase, 10 mg/kg). Anesthesia was induced by intraperitoneal injection of thiopental on days 3 and 4. In the first, second, and third sets of experiments, doses of 40, 60, and 80 mg/kg of thiopental were used, respectively. Results After recording and analysis of data, it became evident that methylene blue owed the most prominent and consistent effect among the chemicals used. After this, nitroglycerine stood on the second order; it potentiated the anesthetic effect of thiopental so that an accelerated anesthesia induction, an increased duration of hypnosis, a prolonged recovery period, and a deeper level of anesthesia were observed. Nitroglycerine, as an NO donor, increased the anesthetic effect of thiopental. This implies that methylene blue should have decreased the anesthesia parameters as it opposes the effect of NO, whereas, similar to what seen with methylene blue, it also increased the duration of anesthesia. Conclusion In conclusion, nitroglycerine, and perhaps other nitrovasodilators, which release NO in physiological environment, may be used as adjuncts to sodium thiopental as an anesthetic agent. The lack of an expected, inhibitory, effect by methylene blue can be related to extra mechanisms implicated in the effects of this chemical.