We have searched for the exclusivity of common sequence motifs of the mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, BMCP1, and plant UCP [PUMP]) within the gene family of mitochondrial anion carrier proteins. The UCP-specific sequences, "UCP signatures", were found in the first, second, and fourth alpha-helices. First: Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-[negatively charged residue]-n/Phe-n/Cys-Thr-Phe/n; second: Gly/Ala-Ile/Leu-Gln/X-[positively charged residue]-NH-n/Cys-Ser/nphi/X-n/Ser-OH/Gly-n-[positively charged residue]-Ile/Met-Gly/Val-n/Thr; fourth: Pro-Asn/ Thr-n-X-[positively charged residue]-Asn/Ser/Ala-n-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-[negatively charged residue]-n-n/Thr/Pro-OH/Val (n, nonpolar; phi, aromatic; (positively charged residue/negatively charged residue, charged residue). The second and part of the third signature are also present in the yeast dicarboxylate transporter. The UCP signature excluding BMCP1 was also found in the second matrix segment: [positively charged residue]-(Pro/ del-Leu/del)-[positively charged residue]-phi-X-Gly/Ser-Thr/n-X-NH/[negatively charged residue]-Ala-phi. These UCP signatures are thought to be involved in fatty acid anion binding and translocation.