Intracytoplasmic sperm injection (ICSI) and partial zona dissection followed by insemination (PZD-I) were used to establish a microinjection system in cynomolgus monkeys (Macaca fascicularis), which are potential models for human reproduction. Two experimental systems were studied, in which either hamster oocytes or cynomolgus monkey oocytes were used as the vehicle. When hamster oocytes were used, 66 out of 81 ICSI-treated oocytes (82%) showed sperm head swelling or pronucleus formation. Following PZD-I of hamster oocytes the rates of spermatozoa penetration (85/114; 75%) and fertilization (71/114; 62%) were relatively high. When cynomolgus monkey oocytes were used, 19 out of 31 (61%) were fertilized by ICSI with cynomolgus monkey spermatozoa and, subsequently, two embryos (7%) developed to the morula stage. In total, 94% (15/16) of the PZD-I treated oocytes were penetrated by spermatozoa and 63% (10/16) were fertilized. These results demonstrate that both micromanipulation techniques can be used in assisted fertilization with cynomolgus monkeys.