A modified method for isolation of rat brain Ca2+, phospholipid-dependent protein kinase has been developed. This procedure enables the purification of the enzyme to a state close to a homogeneous one within 18 hours. The first purification step allows for the separation of two peaks of the Ca2+, phospholipid-dependent protein kinase activity; further purification results in a homogeneous enzyme with a Mr of 80 kDa. It was shown that the second peak of protein kinase is a mixture of two polypeptides with Mr of 80 and 74 kDa. The 74 kDa protein also possesses a catalytic activity and is either an isoform or a proteolytic fragment of the native enzyme. The enzyme activation by phosphatidylserine and phosphatidylinositol was studied. It was shown that in contrast to the phosphatidylserine effect, the dependence of the protein kinase activity on phosphatidylinositol concentration is described by two apparent activation constants, which may be suggestive of the existence of two lipid-binding sites in the enzyme molecule: the enzyme affinity for phosphatidylinositol is 4 times higher than for phosphatidylserine. The data obtained suggest that phosphatidylinositol is a physiological activator of Ca2+, phospholipid-dependent protein kinase.