Recent evidence from this laboratory indicates that in humans there are at least three genes encoding for different isoenzymes of PDK. Here we report data on the enzymatic properties of newly discovered isoenzyme 3, characterized as a recombinant protein, as well as first evidence that the homologous isoenzyme may exist in rodents. Recombinant PDK3 is the most catalytically active isoenzyme characterized thus far. In contrast to isoenzymes 1 and 2, it has very low sensitivity to inhibition by dichloroacetate. the synthetic analog of pyruvate (IC50 of approximately 14 mM), as well as to activation by the products of the pyruvate dehydrogenase reaction - NADH and acetyl-CoA. According to Northern and Western blot analysis in rat tissues, PDK3 isoenzyme is most abundantly expressed in the testis. These observations along with kinetic analysis suggest that PDK3 serves a different physiological function in the regulation of PDH complex then isoenzymes 1 and 2, and explains previous observations on the lack of regulation of testicular PDH by dichloroacetate and fatty acids.