Platelet-activating factor (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine [PAF]) is one of several autocrine trophic factors supporting the development of the preimplantation embryo. PAF acts on the embryo to induce receptor-mediated intracellular calcium (Ca² )_i transients, and these coincide with a marked membrane hyperpolarization. Patch-clamp analysis of 2-cell embryos showed that these Ca² _i transients resulted in an outward membrane current. The present study characterizes this current and assesses its role in embryo development. The outward current was dependent upon the presence of anions in the extracellular medium and occurred as a consequence of the PAF-induced Ca² _i transients. The anion current induced by PAF was inhibited by niflumic acid (NFA), a selective blocker of Ca² -activated Cl^– channels, but this drug did not block the PAF-induced Ca² _i transients. Voltage ramp analysis showed that the Cl^– conductance was outwardly rectifying and inactivated at holding potentials more positive than 30 mV. Culture in NFA or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (a broad-specificity anion channel blocker) from the zygote stage significantly reduced development to blastocysts, with most arresting at the 4-cell and 8-cell stages. Niflumic acid exposure only from the zygote to the late 2-cell stage also reduced the subsequent development to blastocysts. By contrast, treatment from the late 2-cell stage or the 8-cell stage had no effect on development to the blastocyst stage. This study demonstrates the activation of a Ca² -sensitive Cl^– channel in the 2-cell embryo by PAF and shows that this current activity during the zygote to 2-cell stage is required for normal embryo development in vitro.