A technique has been developed for intracytoplasmic sperm injection (ICSI) in the mouse with a relatively low rate of lysis of oocytes (range 5-25% across experiments) and pronuclear formation in around one-third of the intact oocytes (range 30-38% across experiments) for untreated spermatozoa. The treatment of spermatozoa with calcium ionophore, to induce the acrosome reaction (increases acrosome-free spermatozoa from 28 to 58%) before ICSI, increased pronuclear formation to approximately 60% (range 59-62% across experiments) in intact oocytes. The pronuclear oocytes developed to blastocysts in vitro and to term when transferred to recipient foster mothers at rates equivalent to zygotes formed after insemination in vitro. There was no benefit for fertilization rates of activating oocytes with 8% ethanol before or after ICSI, nor was there any evidence of parthenogenetic activation by the sperm solution used for ICSI. This technique adds to other in-vitro fertilization techniques which can be used to explore gamete interactions and to recover breeding in infertile strains and reproductively unfit mice.