The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27°C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4°C, RT, or 37°C (CO₂ incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4°C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at −20°C. Preservation of mouse spermatozoa at 4°C in a medium with high osmolarity (700–1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4°C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at −20°C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.