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We prepared a cell-populated collagen-gel fiber including GbaSM-4 cells derived from the basilar artery of guinea pigs. This fiber tended to be a differentiated contractile phenotype in electron-microscope observations. Sphingosylphosphorylcholine (SPC) can induce contraction of the fiber (EC50 = 0.70 ± 0.05 µM), and blebbistatin can inhibit the SPC-induced contraction (IC50 = 22.8 ± 1.26 µM). Phosphorylation of the 20 kD myosin light chain (MLC20) significantly increased in GbaSM-4 cells provided with 1 µM SPC (P<0.05), which was maintained in the presence of 1 to 100 µM blebbistatin. These results suggest that vascular smooth muscle can relax even if MLC20 is phosphorylated. Keywords:: blebbistatin, sphingosylphosphorylcholine, vascular smooth-muscle cell