Endogenous folate stores are required in preimplantation embryos of several species, but how folates are accumulated and whether they can be replenished has not been determined. Folates are generally taken up into cells by specific transporters, mainly the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptors FOLR1 and FOLR2. Quantitative RT-PCR showed that Slc19a1 mRNA was expressed in mouse cumulus-oocyte complexes (COCs) and oocytes, whereas Folr1 showed expression only in preimplantation embryos, increasing from the 2-cell stage onward. The mRNAs encoding Folr2 and the intestinal folate transporter Slc46a1 were not detected. Methotrexate (MTX), an antifolate often used as a model substrate for folate transport, exhibited saturable transport in COCs and in preimplantation embryos starting at the 2-cell stage. However, folate transport characteristics differed between COCs and embryos. In COCs, transport of MTX and the reduced folate leucovorin was inhibited by the anion transport inhibitor SITS that blocks RFC1 but was insensitive to dynasore, a specific dynamin inhibitor that instead inhibits folate receptor-receptor mediated endocytosis, whereas the opposite was found in 2-cell embryos and blastocysts. The inhibitor profile and transport properties of MTX and leucovorin in COCs correspond to established transport characteristics of RFC1 (SLC19A1), whereas those in 2-cell embryos and blastocysts correspond with those of FOLR1, consistent with the mRNA expression patterns. Considerable folate was accumulated in COCs via RFC1, but the presence of cumulus cells did not enhance folate accumulation in the enclosed oocyte, indicating a lack of transfer from cumulus to oocyte.