The effects of long-term methotrexate (MTX)-induced neutropenia on the periodontal lesion in rats were investigated histologically, histometrically and bacteriologically. A nylon thread was inserted into the interdental gingiva between the 1st and 2nd right maxillary molars of the animals 3 weeks before an application of MTX. The animals were then divided into Groups A and B. Group B were injected intraperitoneally with 1.0 mg/kg of MTX 3 times per week for 9 weeks. Group A received saline as a control. Five animals were killed at the 1st, 3rd, 5th, 7th, and 9th week. In Group A, the neutrophils did not decrease during these 9 weeks. In Group B, however, the neutrophils decreased during the 3rd to 9th week. Whereas the experimental side of Group A showed only moderate alveolar bone resorption between the 7th and 9th week, [in Group B] a marked alveolar bone resorption occurred in Group B occurred in the same period. Alveolar bone loss in the experimental side of Group B was significantly greater (p < 0.01) than in Group A in the 7th to 9th weeks. The percentage of gram-negative rods increased in both control and experimental sides of Group at the 9th week. The results of the present study indicate that neutropenia is induced by a long-term application of MTX in rats and that alveolar bone destruction increases as time goes by in the area where a nylon thread was inserted.