Proton-pumping nicotinamide nucleotide transhydrogenase (Nnt) is a membrane-bound enzyme that catalyzes the reversible reduction of NADP(+) by NADH. This reaction is linked to proton translocation across the membrane. Depending on metabolic conditions, the enzyme may be involved in NADPH generation, e.g., for detoxification of peroxides and/or free radicals and protection from ischemic damage. Nnt exists in most prokaryotes and in animal mitochondria. It is composed of 2-3 subunits in bacteria and of a single polypeptide in mitochondria. An open question is whether Nnt exists in any photosynthetic eukaryotes and if so, to which class it belongs. In the present study it is demonstrated that, by cloning and sequencing cDNA and genomic copies of its NNT gene, an ancient alga, Acetabularia acetabulum (Chlorophyta, Dasycladales), contains a nuclear-encoded Nnt. In contrast to photosynthetic bacteria, this algal Nnt is composed of a single polypeptide of the class found in animal mitochondria. Excluding a poly(A) tail, NNT cDNA from A. acetabulum is 3688 bp long, consists of eight exons and spans 17 kb. The NNT gene from mouse was also characterized. Subsequently, the gene organization of the A. acetabulum NNT was compared to those of the homologous mouse (100 kb and 21 exons) and Caenorhabditis elegans (5.1 kb and 18 exons) genes.