A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1α coding sequence under control of the Polh promoter, plus the mature-E1β coding sequence under control of the p10 promoter. The E1α sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the α- and β-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an α2β2 heterotetramer. Recombinant E1 was able to decarboxylate [1- 1 4 C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.