Different azidocalmodulin derivatives were synthesized by modification of either one carboxylic acid group or one or several arginine residues and their binding and activation capacity investigated in three target enzyme systems. The systems studied were smooth-muscle myosin light-chain kinase, cardiac sarcoplasmic-reticulum kinase and erythrocyte (Mg2+ + Ca2+)-dependent ATPase. The results indicated that the activation ability of each calmodulin derivative was different depending on the system studied. Binding studies carried out by the displacement of 125I-calmodulin indicated that the monosubstitutions did not greatly alter the apparent Kd of calmodulin for the enzymes but that the modification of four arginine residues caused a 4-8-fold increase in the apparent Kd in all systems. These results have shown that azidocalmodulin derivatives may have different degrees of usefulness in the study of calmodulin target proteins in different systems, with the behaviour of the derivatives not predictable on the basis of the nature (soluble or membrane-bound) or the type (ATPase or kinase) of enzyme system to be investigated. However, the monosubstituted calmodulin and, in particular, the carboxylic acid-group-modified derivative (where the modification was statistically dispersed over the protein chain) are good candidates for photolabelling calmodulin-binding proteins.