The two presumed calmodulin antagonists calmidazolium and compound 48/80 were compared for their effects on several calmodulin-dependent and calmodulin-independent enzyme systems. Compound 48/80 and calmidazolium were found to be about equipotent in antagonizing the calmodulin-dependent fraction of brain phosphodiesterase and erythrocyte Ca2+-transporting ATPase. Compound 48/80 combines high potency with high specificity in that: (1) the basal, calmodulin-independent, activity of calmodulin-regulated enzymes was not suppressed; (2) calmodulin-independent enzyme activities, such as Ca2+-transporting ATPases of sarcoplasmic reticulum, Mg2+-dependent ATPases of different tissues and Na+/K+-transporting ATPase of cardiac sarcolemma, were far less altered, or not altered at all, by compound 48/80 as compared with calmidazolium; and (3) antagonism of proteolysis-induced stimulation as opposed to calmodulin-induced activation of erythrocyte Ca2+-transporting ATPase required a 32 times higher concentration of compound 48/80. In all these aspects compound 48/80 was found to be a superior antagonist to calmidazolium since inhibition of calmodulin-independent events by the other agent occurred at considerably lower concentrations. Therefore compound 48/80 is proposed to be a much more specific and useful tool for studying the participation of calmodulin in biological processes than the presently used agents.