Early preantral mouse follicles with a diameter of 110–160 μm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.