Twenty-five 3-acetylcoumarin derivatives were synthesized among which twenty-two were not reported before. Antimutagenic activity screen in vitro has shown that some of these compounds have various activities. The structure and activity relationship for 5-, 7-, 8-substituents has been studied. Pharmacological data showed that: the substituent on position 8 has important effect on its activity. When there is only a hydroxy group on position 7, its activity is the highest among those with other substituents, but when a methyl is on position 8, the order of the activity is reversed. Other trends have also been found which provided some clues for further structural modification.