The dorso-ventral axis in zebrafish first becomes apparent at gastrulation, when the future ventral side appears thinner than the dorsal side. The exact time of establishment of the dorso-ventral axis is not known. We show here that the dorso-ventral axis is specified as early as the 32 cell stage. Using lithium as a marker for dorso-ventral asymmetry, we show that lithium-sensitivity is a characteristic of future ventral cell, but not future dorsal cells, and that there is an asymmetric lithium-sensitivity along the long axis of the 32 cell stage embryo. Consequently, the dorso-ventral axis corresponds to the long axis of the embryo. Because the effect of lithium treatment is short-lived, the dorso-ventral axis must be specified in zebrafish already at the 32 cell stage.