The cyclic AMP-phosphodiesterase assay was used to quantitate the amount of calmodulin activity in various brain areas of male rats treated acutely or chronically for 5 days with morphine. The acute treatment with morphine decreased calmodulin activity in the mitochondrial-synaptosomal P2 fraction of the striatum, midbrain, and thalamus but had no effect on the cerebellum, which contains a low density of opiate receptors. The decrease in calmodulin activity by morphine was dose-dependent and was blocked by the opiate antagonist naloxone. In contrast, chronic treatment of rats with morphine increased calmodulin activity in the mitochondrial-synaptosomal P2 of the striatum, midbrain, cerebral cortex, and thalamus. A highly sensitive Ca2+/Mg2+-ATPase assay was also used to quantitate the amount of calmodulin activity in subcellular fractions obtained from the striatum. Chronic morphine treatment caused a significant increase in calmodulin activity in the membrane containing microsomal, synaptosomal, and mitochondrial layers but only a small change in the layer that contained the soluble proteins and the synaptic vesicles. It is suggested that alteration of the content of calmodulin in specific subcellular sites may have a central role in opiate action and addiction via regulation of multiple calmodulin-sensitive biochemical pathways.