The purpose of this study was to determine the effects of location in the freezing chamber and various freezing protocols on post-thaw development of two-cell mouse embryos. Different protocols were used to cryopreserve two-cell mouse embryos in different freezer positions. Embryos were thawed and cultured to observe post-thaw development. When four different freezer positions were compared, one position provided better post-thaw blastocyst development, regardless of several protocols tested. Furthermore, there were significant differences in temperatures between positions in the freezer. These differences came before seeding (-7 degrees C), just before changing the -7.0 degrees C hold demand (Ramp 2), during the two time points tested during Ramp 3 (-0.3 degrees C/min), and during the holding ramp of -30 degrees C. Post-thaw embryo development was improved by lengthening the time after seeding but before decreasing the temperature again. Decreasing embryos' temperature before plunging appeared to improve post-thaw performance.