Abstract The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A λgt11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity ( T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47–55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5′ and 3′ noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.