Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta, zeta were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.