The mammalian oocyte undergoes two asymmetric cleavages to accomplish meiosis I and II. The actin cytoskeleton is the key player in establishing this asymmetry. Using monoclonal antibodies we have compared the distribution of β- and [upsilon]-cytoplasmic actins (CYAs) in mouse oocytes and early embryos. We show that β-CYA preferentially localized to the cleavage furrow, contractile ring, and cell-cell contact regions. [Upsilon]-CYA showed a widespread cortical localization and was specifically excluded from cell adhesion areas. Oocyte microinjections of isoform-specific perturbators were conducted to modulate specifically and separately isoform functions. Our results suggest that [upsilon]-CYA holds a specific role in the establishment of asymmetry in meiosis I; both β- and [upsilon]-CYA together, appear to participate in the formation and the cortical anchorage of the second meiotic spindle. Our results reveal functional diversity for the two highly conserved non-muscle actin isoforms and provide evidence for an involvement of [upsilon]-CYA in reduced fertility.