We report a cell line (hBSM) established from human urinary bladder wall smooth muscle that maintains most of the phenotypic characteristics of smooth muscle cells. Cells were dissociated from the muscular layer with collagenase (1 mg/ml) and collected and grown in M199 supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic. Primary cultures were grown for 2 d and small colonies were isolated by placing glass rings around the colonies. These colonies were picked up with a fine-tipped Pasteur pipette and subcultured. This procedure was repeated several times until a culture with a uniform stable morphology was obtained. hBSM cells are elongated with tapered ends, and in high density cultures, they form swirls of cells arranged in parallel. These cells have a doubling time of approximately 72 h. Western blotting and immunofluorescence microscopy revealed stable expression of smooth muscle-specific proteins, including myosin isoforms (N-terminal isoforms SM-A/B and C-terminal isoforms SM1/2), SM22, α-smooth muscle actin, h-caldesmon, Ca(2+)-dependent myosin light chain kinase, and protein kinase G. These cells contract upon exposure to 10 μM bethanechol and this contraction is reversible by washing away the drug. Karyotyping showed tetraploidy with a modal chromosome number of 87, with multiple rearrangements. To our knowledge, the hBSM cell line is the first human cell line established from bladder wall smooth muscle that expresses both N- and C-terminal smooth muscle myosin isoforms. This cell line will provide a valuable tool for studying transcriptional regulation of smooth muscle myosin isoforms and effects of drugs on cellular function.