There is clear evidence that the occurrence of specific mRNAs in plasma and serum is associated with cancer, while the usefulness of other body fluids for nucleic acid-based cancer detection remains to be elucidated. Nevertheless, due to the principal advantages of urine (large sample quantities, easy to acquire), several attempts were made to use quantitative reverse transcriptase polymerase chain reaction (RT-PCR)-based detection of putative RNA tumor markers from urine as a tool for noninvasive tumor detection. Because most of the commercially available RNA isolation systems do not accommodate larger sample volumes, the majority of experiments were performed using urine pellets. During the centrifugation step, putative extracellular nucleic acids of low molecular weight as well as complexes containing nucleic acids with low density are lost. Furthermore, cells may be destroyed during this procedure, and the subsequently released nucleic acids will quickly be degraded by nucleases in the urine, which may give rise to inconsistent results. Therefore, we established an improved protocol for the isolation of RNA from urine and subsequent quantification steps. The isolation procedure was tested using a quantitative RT-PCR specific for Ki-67 RNA as well as a radioactive-based reverse transcription approach.