Rim2 is an unusual mitochondrial carrier protein capable of transporting both iron and pyrimidine nucleotides. Here we characterize two point mutations generated in the predicted substrate-binding site, finding that they yield disparate effects on iron and pyrimidine transport. The Rim2 (E248A) mutant was deficient in mitochondrial iron transport activity. By contrast, the Rim2 (K299A) mutant specifically abrogated pyrimidine nucleotide transport and exchange, while leaving iron transport activity largely unaffected. Strikingly, E248A preserved TTP/TTP homoexchange but interfered with TTP/TMP heteroexchange, perhaps because proton coupling was dependent on the E248 acidic residue. Rim2-dependent iron transport was unaffected by pyrimidine nucleotides. Rim2-dependent pyrimidine transport was competed by Zn2+ but not by Fe2+, Fe3+ or Cu2+. The iron and pyrimidine nucleotide transport processes displayed different salt requirements; pyrimidine transport was dependent on the salt content of the buffer whereas iron transport was salt independent. In mitochondria containing Rim2 (E248A), iron proteins were decreased, including aconitase (Fe-S), pyruvate dehydrogenase (lipoic acid containing) and cytochrome c (heme protein). Additionally, the rate of Fe-S cluster synthesis in isolated and intact mitochondria was decreased compared with the K299A mutant, consistent with the impairment of iron-dependent functions in that mutant. In summary, mitochondrial iron transport and pyrimidine transport by Rim2 occur separately and independently. Rim2 could be a bifunctional carrier protein. Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.