PURPOSE: Conventional techniques used to harvest and culture bladder smooth muscle cells (SMCs) have been thought to yield homogeneous populations of SMCs. In order to delineate the cellular composition of tissue derived bladder cells, this study was conducted to determine whether current culturing techniques result in a uniform population of bladder SMCs that may be utilized for bladder tissue engineering. METHODS: Patient derived bladder muscle was isolated and manually minced followed by enzymatic digestion. Cells were cultured in D: -valine alpha-MEM with decreasing levels of fetal bovine serum then fixed and permeabilized for flow cytometric and immunofluorescent analyses. Antibody staining of cultured cells consisted of alpha-SMA, von Willebrand factor, pan-cytokeratin, CD31, and CD90. Cells were visualized using directly conjugated fluorescein isothiocyanate primary or IgG-Alexa-555 conjugated secondary antibodies. RESULTS: Flow cytometric analyses revealed mixed populations of cells expressing non-SMC epitopes as corroborated by immunofluorescent studies. High density oligonucleotide array analysis revealed expression levels of known bladder SMC genes and the expression of endothelial and fibroblast related markers (P < 0.005). CONCLUSIONS: Phenotypic analyses demonstrate cell heterogeneity when SMCs are acquired and cultured through conventional methods. Standardized criteria based upon objective experimentation need to be established in order to better characterize bladder SMCs that are to be utilized for bladder tissue engineering.