The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no difference was observed in the development in vitro between rat oocytes injected with sperm heads separated by sonication or by Piezo-pulse.