Single smooth muscle cells isolated from guinea pig ileum using collagenase and papainproduce contractile response to muscarinic agents, while the cultured cells do not. UsingHuo-3/AM and a confocal laser scanning Huorescence microscope, it was observed thatcarbachol, a muscarinic agent, caused an increase in the intracellular Ca²⁺ of both single andcultured cells. SDS-PAGE and Western Blot analyses revealed the expression of myosinheavy chain isoforms of SM1 (204 kDa) and SM2 (200 kDa) in single smooth muscle cells, andnon-muscle isoform (196 kDa) of myosin heavy chain only in the cultured cells. With respect to actin isoforms, α-actin was predominant in single cells and β-actin was major in the cultured cells. Two types of tropomyosin monomer, 39 kDa and 41 kDa, were detected in single cells, while the 41 kDa monomer was lost in cultured cells. These differences in contractile protein profiles between single and cultured cells were collaborated with the observation of cells using immunofluorescence microscope with responsible antibodies to isoforms of myosin heavy chain, actin and tropomyosin. These results suggest that the loss of contractility in cultured smooth muscle cells is profoundly related to changes in contractile protein profiles from smooth muscle type to non-muscle type.