Mutants were constructed for mitochondrial uncoupling protein UCP1, with single or multiple substitutions within or nearby the UCP-signatures located in the first alpha-helix and second matrix-segment, using the QuickChange site directed mutagenesis protocol (Stratagene), and were assayed fluorometrically for kinetics of fatty acid (FA)-induced H+ uniport and for Cl- uniport. Their ability to bind 3H-GTP was also evaluated. The wild type UCP1 was associated with the FA-induced H+ uniport proportional to the added protein with a Km for lauric acid of 43 micro M and Vmax of 18 micro molmin(-1)(mg protein)(-1). Neutralization of Arg152 (in the second matrix-segment UCP-signature) led to approximately 50% reduction of FA affinity (reciprocal Km) and of Vmax for FA-induced H+ uniport. Halved FA affinity and 70% reduction of Vmax was found for the double His substitution outside the signature (H145L and H147L mutant). Neutralization of Asp27 in the first alpha-helix UCP-signature (D27V mutant) resulted in 75% reduction of FA affinity and approximately 50% reduction of Vmax, whereas the triple C24A and D27V and T30A mutant was fully non-functional (Vmax reduced by 90%). Interestingly, the T30A mutant exhibited only the approximately 50% reduced FA affinity but not Vmax. Cl- uniport and 3H-GTP binding were preserved in all studied mutants. We conclude that amino acid residues of the first alpha-helix UCP signature may be required to hold the intact UCP1 transport conformation. This could be valid also for the positive charge of Arg152 (second matrix-segment UCP signature), which may alternatively mediate FA interaction with the native protein.