The inhibition of sarcoplasmic reticulum Ca²⁺-ATPase activity by miconazole was dependent on the concentration of ATP and membrane protein. Half-maximal inhibition was observed at 12 µM miconazole when the ATP concentration was 50 µM and the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a low micromolar concentration of miconazole activated the enzyme, whereas higher concentrations inhibited it. A qualitatively similar response was observed when Ca²⁺ transport was measured. Likewise, the half-maximal inhibition value was higher when the membrane concentration was raised. Phosphorylation studies carried out after sample preequilibration in different experimental settings shed light on key partial reactions such as Ca²⁺ binding and ATP phosphorylation. The miconazole effect on Ca²⁺-ATPase activity can be attributed to stabilization of the Ca²⁺-free enzyme conformation giving rise to a decrease in the rate of the Ca²⁺ binding transition. The phosphoryl transfer reaction was not affected by miconazole.