Introduction: Both initiation and maintenance of spermatogenesis are hormonally regulated by FSH and testosterone. In this study, the effects of FSH and testosterone on the in vitro maturation of fresh and frozen- thawed round spermatids were determind. Material and Methods: Cell suspension was isolated from testis of NMRI male mice (8-10 weaks old) and divided into two groups .The first part was used to culture freshly and the second part quickly cryopreserved using 18% raffinose (w/v) and 3% skim milk (w/v) in distilled water. Each part of cell suspension was divided into two groups: the fresh specimen was cultured in medium containing DMEM and 10% FBS and medium supplemented with FSH (50 IU/L) and testosterone (1µmol/l). The frozen -thawed specimen cultured on DMEM & 10% FBS and DMEM & 10% FBS was supplemented with rFSH & testosterone. The number of round, elongating and elongated spermatids, before and during culture were counted up daily for 96 h using light microscope. Survival rates of all kinds of spermatids were evaluated using trypan blue test and the results of every group were compared statistically by a repeated measure ANOVA. Results: The results of this study showed that in fresh specimen cultured in medium suppelemented with FSH and testosterone, on the first 24h, the numbers of round spermatid cells were reduced but elongating and elongated spermatid cells increased. Between 24 to 96h, all kinds of spermatid cells were reduced .In frozen-thawed group cultured in medium supplemented with FSH & testosterone, although there was a severe reduction in the number of round spermatids, the number of elongating and elongated cells did not change during the first day of culture. Viability rates of all kinds of spermatid cells reduced during 96h culture in the both fresh and frozen-thawed specimens. Conclusion: Results of this study confirm that round spermatid cells can progress into elongating and elongated cells only over the first 24h culture period in fresh and frozen-thawed specimen cultured in medium supplemented with FSH and testosterone.