The dicarboxylate carrier is an important member of the mitochondrial carrier family, which catalyzes an electroneutral exchange across the inner mitochondrial membrane of dicarboxylates for inorganic phosphate and certain sulfur-containing compounds. Screening of the Drosophila melanogaster genome revealed the presence of a mitochondrial carrier subfamily constituted by four potential homologs of mammalian and yeast mitochondrial dicarboxylate carriers designated as DmDic1p, DmDic2p, DmDic3p, and DmDic4p. In this paper, we report that DmDIC1 is broadly expressed at comparable levels in all development stages investigated whereas DmDIC3 and DmDIC4 are expressed only in the pupal stage, no transcripts are detectable for DmDIC2. All expressed proteins are localized in mitochondria. The transport activity of DmDic1-3-4 proteins has been investigated by reconstitution of recombinant purified protein into liposomes. DmDic1p is a typical dicarboxylate carrier showing similar substrate specificity and inhibitor sensitivity as mammalian and yeast mitochondrial dicarboxylate carriers. DmDic3p seems to be an atypical dicarboxylate carrier being able to transport only inorganic phosphate and certain sulfur-containing compounds. No transport activity was observed for DmDic4p. The biochemical results have been supported at molecular level by computing the protein structures and by structural alignments. All together these results indicate that D. melanogaster dicarboxylate carriers form a protein subfamily but the modifications in the amino acids sequences are indicative of specialized functions.