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1. ATP γP-imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+-ATPase of sarcoplasmic reticulum in a biphasic manner. A fast first phase is concentration-dependent while a slower second phase is independent of the inhibitor concentration. 2. The inhibition is calcium- and magnesium-dependent. No inhibition occurs in the absence of either cation. 3. Inhibition of the Ca2+-ATPase can be prevented by the protection with ATP. 4. The loss of ATPase activity is pH-dependent. Maximal inhibition coincides with maximal ATPase activity. It indicates a participation of the reacting amino acid side chain in the catalytic cycle. 5. The incorporation of radioactive inhibitor is reversed in a time-dependent fashion while the Ca2+-ATPase remains inhibited. 6. We conclude that ATP γP-imidazolidate initially reacts covalently with an amino acid side chain, probably Asp-351, but is subsequently expelled by a reaction with a second amino acid. 7. This two-step reaction induces an intramolecular cross-link which can be shown by the creation of a new protein band on SDS-PAGE which originates in the Ca2+-ATPase. [ABSTRACT FROM AUTHOR]