Introduction: The histopathology and expression of nucleolar organizing regions (NORs), of seminiferous tubules was studied after the administration of methotrexate (MTX) and vitamin A (VA). Objective of the study was to test if the NOR count increases or decreases with different doses of MTX. Material and Methods: Male Wistar rats aged 4 months, maintained in our institution were used in the present study. The rats were divided into following groups, group I – control (saline treated), group II – animals treated with 8 mg of MTX, group III – animals treated with 10 mg of MTX, group IV – animals treated with 12 mg of MTX, group V - animals treated with VA (5000 IU), Group VI – animals treated with VA (5000 IU) and MTX (12 mg). Results: The animals treated with MTX showed significant decrease in the diameter of seminiferous tubules and increase in interstitial space. The spermatogenesis was not complete in some tubules. When MTX was given with VA, the damage done by MTX was decreased. There was an overall numerical decrease in the total NOR count from the control to 12 mg dose MTX. The total count included small, medium and large sized NORs. Conclusion: Higher the dose of MTX, lesser the NOR count and lesser the irregularity of NORs. However VA co-administration reduces the damage produced by MTX. We believe that findings of the present study might be useful in understanding the long term effect of MTX and its adverse effects.