To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.