The large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase (LCL), situated between Lys329 and Phe740, is believed to contain both its phosphorylation and ATP binding domains. A cDNA fragment coding for this amino acid sequence was generated in vitro and cloned in vector pQE8 which allowed the overexpression in Escherichia coli of this Ca(2+)-ATPase domain fused with a cluster of 6 histidines at its NH2 terminus. The fusion protein produced in an insoluble form within bacteria was solubilized in 4 M urea, purified on immobilized Ni2+, and then renatured by elimination of urea. More than 4 mg of purified renatured fusion protein was obtained from 500 ml of culture. ATP binding on the refolded protein was demonstrated by two methods: 1) detection of ATP-induced intrinsic fluorescence change and 2) binding of the fluorescent ATP analogue 2',3'-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP) and its chase by ATP. It is shown that the LCL protein has one single TNP-ATP binding site having a dissociation constant (Kd) of 1.6-1.9 microM. Both methods yielded a Kd for ATP around 200 microM. Binding of other nucleotides was detected with a sequence of Kd identical to that found for native Ca(2+)-ATPase: ATP < ADP < GTP < AMP < ITP. A Mg2+ binding site was also found on the LCL protein (Kd = 100 microM at pH 7.2). The fluorescence of bound TNP-ATP was found to be highly dependent on Mg2+ binding on this site.