This review attempts to provide a comprehensive overview of the biology of the physiologically and pharmacologically important transport system termed the "reduced folate carrier" (RFC). The ubiquitously expressed RFC has unequivocally established itself as the major transport system in mammalian cells and tissues for a group of compounds including folate cofactors and classical antifolate therapeutics. Loss of RFC expression or function may have potentially profound pathophysiologic consequences including cancer. For chemotherapeutic antifolates used for cancer such as methotrexate or pemetrexed, synthesis of mutant RFCs or loss of RFC transcripts and proteins results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and insufficient substrate for polyglutamate synthesis. Since RFC was first cloned in 1994, tremendous advances have been made in understanding the complex transcriptional and posttranscriptional regulation of RFC, in identifying structurally and functionally important domains and amino acids in the RFC molecule as a prelude to establishing the mechanism of transport, and in characterizing the molecular defects in RFC associated with loss of transport in antifolate resistant cell line models. Many of the insights gained from laboratory models of RFC portend opportunities for modulating carrier expression in drug resistant tumors, and for designing a new generation of agents with improved transport by RFC or substantially enhanced transport by other folate transporters over RFC. Many of the advances in the basic biology of RFC in cell line models are now being directly applied to human cancers in the clinical setting, most notably pediatric acute lymphoblastic leukemia and osteogenic sarcoma.