Double-reciprocal plots of the rate of ATP hydrolysis by Na+,K(+)-ATPase versus ATP concentration are not linear, and may reflect either two distinct binding sites for ATP or a single ATP binding site whose affinity for the nucleotide alternates between high-affinity and low-affinity states. In order to determine whether multiple nucleotides or nucleotide analogs can bind simultaneously to Na,+,K(+)-ATPase, the effects of nucleotides on the hydrolysis of p-nitrophenyl phosphate and on the dephosphorylation rate of Na+,K(+)-ATPase modified by fluorescein 5'-isothiocyanate (FITC) were measured. FITC blocks the high-affinity binding site for ATP on the Na+K(+)-ATPase and inhibits ATP hydrolysis at ATP concentrations as high as 8.3 mM. The hydrolysis of p-nitrophenyl phosphate and phosphoenzyme formation from inorganic phosphate and Mg2+ were not affected by FITC modification. The p-nitrophenylphosphatase activity of unmodified Na+,K(+)-ATPase was stimulated by low concentrations of ATP (10-100 microM) and other nucleotides, and was inhibited at higher nucleotide concentrations. In contrast, there was no effect on p-nitrophenyl phosphate hydrolysis by FITC-modified Na,K(+)-ATPase at ATP concentrations less than 100 microM. The hydrolysis of p-nitrophenyl phosphate by FITC-modified Na+,K(+)-ATPase was inhibited at ATP concentrations greater than 100 microM. These observations demonstrate that the effects of ATP acting at high-affinity sites are absent in FITC-modified Na+,K(+)-ATPase but the effects of ATP acting at low-affinity sites are still observed. In unmodified Na+,K(+)-ATPase, the rate of dephosphorylation of the phosphoenzyme formed from inorganic phosphate and Mg2+ was inhibited by ATP.(ABSTRACT TRUNCATED AT 250 WORDS)