Calponin, an actin-linked regulatory protein in smooth muscle, caused a remarkable change in the fluorescence intensity of pyrene-labeled actin in the filamentous form. Calponin, an equimolar ratio to actin, decreased the fluorescence intensity of pyrene-labeled F-actin by some 60% to the level near monomeric actin. This change was partially reversed by Ca2+, when calmodulin was present. Thus it appears that calponin causes conformational changes in actin molecules in an actin filament so as to inhibit their interactions with myosin.