Aims: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. Methods and Results: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. Conclusions: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. Significance and Impact of Study: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.