Bacteria capable of denitrification play a significant role in the nitrogen cycle of freshwater ecosystems. By metabolizing nitrogen compounds they e.g. counteract the eutrophication of natural waters. To get detailed insights into the in situ turnover rates of nitrogen a reliable tool of quantification for active microorganisms is essential. In the present investigation, quantification capabilities of a molecular tool (Polymerase Chain Reaction—PCR) and a cultivation based tool (Most probable number—MPN) were investigated and compared. The total bacterial concentration yielded by the molecular PCR approach was up to 6-fold higher compared to the results of the MPN approach. However, the portion of culturable denitrifying bacteria compared to the number of specific gene copies (nirS) was much lower. Depending on the environmental conditions, the difference between the PCR and the MPN approach was up to three orders of magnitude. From lab scale experiments with a pure P. aeroginosa strain it can be concludes, that these differences are not the result of inappropriate culture conditions but rather reflect the portion of so called viable but not culturable bacteria (VBNC). Low nitrate concentrations as found in many fresh water ecosystems induced a significant increase in the portion of non culturable denitrifying bacteria. Referred to the investigation of dynamic populations, the number of metabolic active bacteria is represented by the MPN rather than by the PCR approach.