For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca(2+) oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.