The alpha subunit of the mitochondrial ATP synthase is part of the F1 enzymatic complex known to bind ADP, phosphate and ATP and is at the heart of the mitochondrial energy-producing mechanism. The mouse embryonal carcinoma variant of the alpha subunit cDNA was cloned and the complete nucleotide sequences of two different lengths of clones were determined. Two distinct polyadenylation sites in the cDNA sequence were detected and two sizes of mRNAs were confirmed by Northern blot hybridization. Two putative ATP-binding motifs - A and B, have been hypothesized for this enzyme based on previous NMR work on another ATP-binding enzyme, adenylate kinase. We have constructed four deletion mutants of the alpha subunit of the mouse F1-ATP synthase to examine the putative role of these domains. The resulting recombinant proteins were expressed and purified. Functional studies with the immobilized mutants proved the significance of both sites for ATP binding.