In 1990, the New York City Department of Environmental Protection (DEP) began investigating a seasonal increase in the fecal coliform concentrations in Kensico Reservoir, Valhalla, New York. This location is significant since Kensico Reservoir is the last storage reservoir prior to chlorination for approximately 80-90% of the drinking water that is delivered to nine million New York City residents daily. This study, coupled with the new coliform regulations mandated by the Surface Water Treatment Rule (SWTR), required a new approach for the way DEP microbiologists would respond to all future investigations. The DEP formed a Research Microbiology Unit that would work with the routine microbiology staff to provide more in-depth investigations into unusual occurrences in the upstate watershed. The main function of this group has been to determine if fecal contamination, as suggested by microbial indicator analyses, is coming from human or non-human sources. This approach has helped to narrow down sources of fecal input in the watershed, and allowed preventative measures to be taken to greatly reduce or eliminate further contamination. In addition, the specificity of some of this work has also helped to assess the human infective potential of samples in some situations. This has been of great value when determining potential health risks to consumers. The routine microbial indicators used by the Department have been total coliform, fecal coliform, Escherichia coli, Giardia spp. and Cryptosporidium spp. Follow up analysis used to narrow down sources of fecal contamination have included: fecal streptococci, enterococci, FC:FS ratios, serotyping, electrophoretic typing, F-specific RNA coliphage typing, Ribotyping (RT), Small Sub-Unit (SSU) rRNA based Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) and gene sequencing. The level of confidence associated with each of these techniques is unique to each investigation and has also been dependant upon other water quality data and supplemental information provided. In studies where multiple techniques have been applied, and have been in agreement, the confidence in the conclusion has been greatly increased. Some of these techniques have not performed as well as others, and some have only appeared to be of benefit when used in combination with other techniques. Each investigation has been somewhat distinctive and has required a certain method, or combination of techniques, to reach a result. Conversely, there have been times when applied methods have not been helpful in making a source determination and no conclusions could be made. Consequently, the DEP continues to investigate and develop methods to become part of its “microbial toolbox”, and there are continued plans for future work in the science of source tracking