Negatively charged amino acids, such as aspartate and glutamate, were selected as substrates by low- and high-Km components of mediated Na(+)-dependent transport in preimplantation mouse blastocysts. These and other relatively small anionic amino acids with two carbon atoms between the negatively charged groups (or up to three carbon atoms when the groups were both carboxyl groups) interacted strongly with the low-Km component of transport, whereas larger anionic amino acids interacted weakly or not at all. The low-Km system was also stereoselective except in the case of aspartate. Moreover, transport was Cl(-)-dependent and slower at pH values outside the range 5.6-7.4. L-Aspartate, D-aspartate and L-glutamate each interacted strongly with the low-Km component of transport with Km values for transport nearly equal to their Ki values for inhibition of transport of one of the other amino acids. By these criteria, the low-Km component of transport of anionic amino acids in blastocysts appears to be the same as the familiar system X-AG that is present in other types of mammalian cells. In contrast, the high-Km component of transport in blastocysts preferred L-aspartate to L-glutamate, whereas the reverse is true for fibroblasts. Therefore, transport of anionic amino acids in blastocysts may occur via at least one process that has not been described in other types of cells. Roughly half of mediated glutamate and aspartate transport in blastocysts may occur via the high-Km component of transport at the concentrations of these amino acids that may be present in uterine secretions.