In recent years, the phenomena of bacterial regrowth in water distribution systems has been identified at an ever increasing rate. The problem has appeared and disappeared in numerous distribution systems without verifiable explanations as to cause. This paper details Detroit's experience in the investigation of the regrowth phenomena. It details the problems encountered in entering a large water main in preparation for bacteriological sampling and details the techniques employed to conduct bacteriological and biofilm sampling while minimizing contamination. The paper then presents the findings of this unique examination of an in-service water main for biofilm formation and biological components in the water distribution system. It was later determined that the most probable cause of the bacteriological growth was due to a cross connection in the system.