Fesselin is a basic protein isolated from smooth muscle which binds G-actin and accelerates its polymerization as well as crosslinks assembled filaments [J. Muscle Res. Cell Motil. 20 (1999) 539; Biochemistry 40 (2001) 14252]. In this report experimental evidence is provided for the first time proving that fesselin can interact with calmodulin in a Ca2+-dependent manner in vitro. Using ion exchange, followed by calmodulin-affinity chromatography, enabled us to simplify and shorten the fesselin preparation procedure and increase its yield by about three times in comparison to the procedure described by Leinweber et al. [J. Muscle Res. Cell Motil. 20 (1999) 539]. Fesselin interaction with dansyl-labelled calmodulin causes a 2-fold increase in maximum fluorescence intensity of the fluorophore and a 21 nm blue shift of the spectrum. The transition of complex formation between fesselin and calmodulin occurs at submicromolar concentration of calcium ions. The dissociation constant of fesselin Ca2+/calmodulin complexes amounted to 10(-8) M. The results suggest the existence of a direct link between Ca2+/calmodulin and fesselin at the level of actin cytoskeleton dynamics in smooth muscle. (C) 2004 Elsevier Inc. All rights reserved.