The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5 % CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 mu M) and cultured individually for 7 days on Vero cells in microdrops. In seven trials, 353 cumulus-oocyte complexes were matured and 103 oocytes were microinjected. Eight oocytes were sham microinjected. After ICSI, 85 oocytes (82.5 %) survived the sperm injection procedure. Among the 76 successfully microinjected oocytes, 52 (68 %) were fertilized (two pronuclei, syngamy stage and cleaved oval. Sham microinjected oocytes were not activated. After in vitro culture, 35 ova (46 %) were cleaved 2 days after ICSI and early embryonic development was obtained (three embryos of 23 cells, 50 cells and more than 80 cells) 5 to 7 days after ICSI