Purpose: In our previous study, morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells were seen immediately after 7-day testosterone treatment (T group) in rat testes. The results strongly suggested that significant regulatory factors in spermatogenesis remain to be discovered. The present study was conducted to determine the morphometric and ultrastructural analysis of rat sertoli and spermatogenic cells in stage IX T group in one group and the same treatment in another group followed by 7-day non treatment (AT group), and also to assess whether or not the suggested unknown regulatory factors exist in the AT group. Results: In the AT group, concentrations of testosterone decreased more prominently than in its counterpart in the T group. However, its concentration was lower than its counterpart in the control group of rats. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the AT group increased significantly more than their counterparts in the T group. Further they were closer to their counterparts in the control group than their counterparts in the T group. The absolute volumes of seminiferous tubules in the AT group increased to levels closer to their counterparts in the control group than to their counterpart in the T group. In the cytoplasm of the Step 9 spermatid in the T group, disorganization of the microtubules in a manchette-like structure appeared. However, they assumed a normal manchette-like orientation in the AT group. The fine structures corresponding to the transverse section of tails in normal spermatozoa were detected in the adluminal region of the seminiferous epithelium in the AT group, and these findings suggest that normal spermatozoa were formed in the AT group. Conclusion: The present study strongly suggests that unknown regulatory factors remain to be discovered.