Factors affecting full-term development of rat oocytes after round spermatid injection (ROSI) were examined using fresh and cryopreserved spermatids. Regardless of method (DC pulses or ionomycin) and timing (before or after ROSI) for oocyte activation, similar percentages of oocytes injected with cryopreserved spermatids developed to full-term (3.0-4.8%). In contrast, no offspring were obtained when fresh spermatids were injected into DC-treated oocytes (0%), but a higher proportion of oocytes first injected with fresh spermatids and then activated with ionomycin developed to full-term (6.1%). In further experiment, oocytes activated with ionomycin were injected with frozen-thawed spermatids, and then treated with cycloheximide and/or trichostatin-A. Neither chemical had a beneficial effect on full-term development of ROSI oocytes (3.7-7.9% vs 2.5% in control). In conclusion, regardless of timing of oocyte activation, activation treatment with ionomycin is required for full-term development of rat oocytes injected with fresh spermatids, and the higher potential of cryopreserved spermatids contributing to full-term development is notable when DC pulse is applied for oocyte activation.