When intracytoplasmic sperm injection (ICSI) is applied in the rat, sperm chromatin is introduced into the oocyte together with the acrosome, which does not enter the cytoplasm of the oocyte during normal fertilization, resulting in the rat giving birth to pups. Since successful ICSI was reported in rats, but with low efficiency, it has been observed that the acrosome of the sperm head seems to have detrimental effects on the embryonic development of ICSI oocytes. To improve ICSI in rats, the effects of removal of the acrosomal membrane from rat sperm on the development of ICSI oocytes were examined. While most control (non-treated) sperm had an intact acrosomal membrane, the Triton X-100 (TX)- and lysolecithin (LL)-treated groups showed high percentages of sperm with a removed acrosomal membrane. The timing of pronuclear formation in ICSI-oocytes using TX- or LL-treated sperm was significantly accelerated compared with that of the control sperm (P<0.05). However, neither TX nor LL treatment affected amounts of PLCζ in rat sperm. The rates of offspring derived from TX- (20.3 ± 4.4%) and LL-treated sperm (19.0 ± 2.8%) were also significantly higher than that of the control group (7.6 ± 2.3%; P<0.05). Our data clearly indicate that removal of acrosomal membranes from sperm by reagents is effective for generation of offspring via ICSI in rats.