We have investigated the role of Hsp27 phosphorylation and association of Hsp27 with the contractile proteins actin, myosin and tropomyosin. Smooth muscle cells were labeled with [³²P]-orthophosphate. C₂ Ceramide (0.1 µM) an activator of protein kinase C induced a sustained increase in Hsp27 phosphorylation that was inhibited by calphostin C. C₂ ceramide-induced (0.1 µM) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of Hsp27 with tropomyosin and in the association of Hsp27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of Hsp27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin and Hsp27, followed by confocal microscopy. Transfection of smooth muscle cells with Hsp27 phosphorylation mutants indicated that phosphorylation of Hsp27 could affect myosin association with actin, possibly through Hsp27 association with tropomyosin. In conclusion: 1) Hsp27 phosphorylation appears to be necessary for reorganization of Hsp27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway; 2) agonist-induced phosphorylation of Hsp27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin.