The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-Å crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic α-helices, whereas the C-terminal half is folded into an α/β sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.