In the rat micronucleus test, it was reported that phenacetin induced micronuclei in peripheral blood, but not in bone marrow. In this study, we used the new methylene blue/acridine orange (N/AO) staining method and the acridine orange (AO) staining method to examine whether phenacetin induces micronuclei in rat bone marrow. Utilizing the method of N/AO staining, reticulocytes were classified into Type I to IV, and then micronucleated reticulocytes (MNRETs) of each type were counted. Micronucleated polychromatic erythrocytes (MNPCEs) were detected in AO-stained specimens. After a single oral administration of phenacetin, MNRETs were mainly increased in Type II and Type III reticulocytes at 24 and 48 hours, respectively, but were not increased at 72 h after administration at a dose of 2000 mg/kg. In addition, the frequency of MNRETs in reticulocytes of Type I and Type II was increased at doses of 500, 1000 and 2000 mg/kg, and the MNRETs frequency at 2000 mg/kg was 3-fold higher than that of MNPCEs at the same dose. After two or four repeated doses of phenacetin, MNRETs were mainly increased in both Type II and Type III reticulocytes at 24 h after final administration. Similar magnitude increases in the frequencies of MNRETs in Type I and Type II reticulocytes, and in Type III reticulocytes and MNPCEs were found for both two and four repeated doses. These results indicate that phenacetin can induce micronuclei in rat bone marrow and that the N/AO staining method in combination with micronucleus scoring in the restricted type(s) of reticulocytes is useful in the rat micronucleus test.