Compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 48/80 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 micrograms/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 micrograms/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound 48/80 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound 48/80 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 48/80 bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 48/80 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound 48/80 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 48/80, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound 48/80 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 48/80 is proposed to be a promising tool for studying calmodulin-dependent processes.