Background Bladder cancer, the 5th most common malignancy in the USA, is often detected as a result of incidental findings or by presenting hematuria. Once diagnosed the disease is one of the costliest cancers to treat due to frequent, invasive and often lifelong follow-up procedures. Because cells are shed into urine, there has been an emerging effort to develop non-invasive tests for the detection of bladder cancer. Expression of survivin, a member of the inhibitor of apoptosis protein family, has been associated with bladder cancer. Therefore, the goal of this study was to determine the feasibility of transducing viable exfoliated cells obtained from urine with an adenoviral vector in which a reporter gene is under the control of the survivin promoter. Methods Exfoliated cells from urine were obtained from 36 human subjects (> 40 years old). An adenovirus in which GFP expression is under control of the survivin promoter (Ad.Surv.GFP) was generated. An adenovirus in which GFP is expressed from the CMV promoter served as a control. GFP expression was analyzed by fluorescent microscopy and quantified by flow cytometry. Results Short-term cultures from exfoliated cells in urine could be established in 16 of 31 samples. These cultures were successfully transduced with Ad.CMV.GFP. Analysis of GFP expression following transduction with Ad.Surv.GFP, indicated that the survivin promoter was preferentially active in UM-UC-3 bladder cancer cells compared to non-malignant UROtsa cells. Interestingly, baseline levels of GFP expression in cultures from exfoliated cells in urine exhibited higher baseline levels than UROtsa following transduction with Ad.Surv.GFP. Conclusions We demonstrated the feasibility of establishing and analysing short-term cultures isolated from exfoliated cells in voided urine by means of adenoviral transduction, thereby forming the foundation for future studies to determine the specificity and sensitivity of a non-invasive test based on survivin promoter activity.