The segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence. Once folded, DN alpha 19 was highly soluble and stable. It bound 1 mol/mol either of adenine or guanine di- or triphosphate nucleotide, with a Kd ranging from 2.3 to 5.4 microM, or of methylanthraniloyl derivatives of the same nucleotides, with a Kd ranging from 0.2 to 0.6 microM. Interesting, DN alpha 19 was able to hydrolyze nucleoside triphosphates at a low but significant rate. The distance between one tryptophan residue located in the nucleotide-binding site and the ribose-linked methylanthraniloyl group of di- or triphosphate nucleotides was estimated by fluorescence resonance energy transfer to be 13 or 11 A, respectively, suggesting that the tryptophan is close to the polyphosphate moiety of the nucleotide. This tryptophan residue was tentatively assigned to W190 by a hydrophobic cluster comparison with the H-ras p21 protein, suggesting that the putative loop of DN alpha 19 containing W190 could play a functional role in nucleotide binding.