Activity-guided fractionation of methanol:chloroform (1:1) extract of Zataria multiflora Boiss was performed in order to investigate the active component(s) responsible for antinociceptive activity. Antinociceptive activity was evaluated using hot-plate and writhing tests. Morphine (10mg/kg, i.p.) and diclofenac (10mg/kg, i.p.) were used as positive controls. The methanol-chloroform (1:1) extract of the aerial parts of the plants was prepared and partitioned between methanol-water (9:1) and n-hexane. The more active hydroalcoholic fraction was concentrated and further partitioned between methanol-water (3:2) and chloroform. The hydroalcoholic layer showed a dose-dependent and significant activity compared to morphine and diclofenac. Further fractionation of the hydroalcoholic fraction on silica gel gave six fractions. Three fractions at 100mg/kg showed activity comparable to those of morphine.