The purpose of this study was to probe the regulatory nucleotide site of the Ca2+-ATPase of sarcoplasmic reticulum and to study its relationship with the catalytic nucleotide site. Our approach was to use the nucleotide analogue 2'(3')-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-phosphate (TNP-AMP), which is known to bind the Ca2+-ATPase with high affinity and to undergo a manyfold increase in fluorescence upon enzyme phosphorylation with ATP in the presence of Ca2+. TNP-AMP was shown to bind the regulatory site in that it competitively inhibited (Ki = 0.6 microM) the secondary activation of turnover induced by millimolar ATP, thus providing a high affinity probe for the site. Observation of the high phosphoenzyme-dependent fluorescence upon monomerization of the enzyme without an increase in phosphoenzyme levels showed the regulatory site to be on the same subunit as the catalytic site and excluded an uncovering of "silent" nucleotide sites resulting from dissociation of enzyme subunits. Identical stoichiometric levels of [3H]TNP-AMP binding (4 nmol/mg of protein) to either the free enzyme or the enzyme phosphorylated with 250 microM ATP excluded models of two nucleotide sites per subunit. Finally, transient kinetic experiments in which TNP-AMP was found to block the ADP-induced burst of phosphoenzyme decomposition showed that TNP-AMP was bound to the phosphorylated catalytic site. We conclude that the regulatory nucleotide site is not a separate and distinct site on the Ca2+-ATPase but, rather, results from the nucleotide catalytic site following formation of the phosphorylated enzyme intermediate.