Mammals lack de novo folate synthesis pathways and rely on the uptake of folate from the cell exterior through membrane transporters such as the human reduced folate carrier (hRFC) and the proton-coupled folate transporter (PCFT). Many cancers show disruptions in folate metabolism, and antifolate drugs that target these transporters — such as methotrexate (MTX) and pemetrexed (PMX) — are effective treatments for a range of cancer types. As different antifolates show different target specificity, understanding the binding sites of these drugs could help with the rational design of therapeutics that selectively target specific folate transporters. Writing in Nature, Wright et al. now elegantly profile the binding of MTX to hRFC. Cryo-EM structures showed that hRFC adopts an MFS fold, comprising 12 transmembrane helices that produce a spacious binding pocket capable of accommodating a variety of antifolates and reduced folates. By modifying amino acids in part of the binding pocket proximal to the head group of bound antifolate drugs, the authors found that they could modify the preference of hRFC for different substrates — thus also outlining how the modification of antifolates could tune their specificity to hRFC. Molecular dynamics simulations and a comparison of the MTX–hRFC structure with the recently described structure of PMX-bound PCFT implicated other aspects of antifolates that govern their specificity, including the length of their linker regions. Further work will interrogate alternative conformations of drug-bound folate carriers to better understand the structural features that determine the specificity of antifolate drugs.