R-24571 (calmidazolium), a derivative of the antimycotic agent miconazole, inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), with an IC50 (the concentration causing 50% inhibition) of 5.3 microM. It also inhibited the calmodulin/Ca2+-stimulated enzymes, with IC50 values of 1.6 and 0.1 microM for myosin light chain kinase (MLCK) and phosphodiesterase respectively. Analysis of inhibition by R-24571 of PL-Ca-PK and MLCK revealed complex kinetics, suggesting that the agent interacted with the cofactors, the enzyme, and/or the cofactor-enzyme complexes. At saturating concentrations of the cofactors, R-24571 inhibited PL-Ca-PK and MLCK noncompetitively with their respective cofactors. Inhibition of MLCK by R-24571 was completely overcome by phosphatidylserine, indicating a strong hydrophobic interaction between R-24571 and the phospholipid in the presence of calmodulin. R-24571 also inhibited phosphorylation of various endogenous proteins in brain stimulated specifically by phosphatidylserine/Ca2+ or calmodulin/Ca2+. The present findings inducated that R-24571 has little specificity in inhibiting two types of Ca2+-dependent protein kinases sensitive to phospholipid or calmodulin.