Rat sperm freeze-dried in a solution containing Tris and ethylenediaminetetraacetic acid (EDTA) (TE buffer) can be preserved at 4 degrees C, and oocytes injected with these sperm developed into offspring though developmental ability was low. We studied the culture conditions to improve the developmental ability of oocytes injected with freeze-dried sperm. After being injected with fresh sperm, the zygotes were cultured in modified Krebs-Ringer bicarbonate (mKRB), modified rat 1-cell embryo culture medium (mR1ECM)/BSA, and mR1ECM with different osmolality, before being cultured in mR1ECM. High proportion of zygotes cultured in mKRB (270mOsm) before being cultured in mR1ECM developed into blastocysts compared to zygotes cultured only with mR1ECM (50% vs. 28%, P<0.05). Culturing in mKRB also led to a high proportion of zygotes developing into blastocysts after the injection of freeze-dried sperm than zygotes cultured only with mR1ECM (32% vs. 15%, P<0.05). Offspring (16%) were obtained when 19 2-cell embryos derived from oocytes that had been injected with freeze-dried sperm preserved at 4 degrees C for 1year were transferred. This study demonstrated that the culture conditions soon after the injection of sperm markedly influenced the subsequent development of embryos. Also, rat sperm after freeze-drying in TE buffer were preserved at 4 degrees C for long term without their deterioration.