The Autoanalysis Colilert (AC) test is a modification of the defined substrate technology designed to enumerate specific target microbe(s) from a mixture. All ingredients are present in the tube or vessel in powdered form. The AC test is simple to perform—a measured amount of water is added to test tubes (for the most probable number test) or vessel [Presence/Absence (P/A) test]. After incubation, the development of a yellow color is specific for total coliforms. Fluorescence in the same tube(s) is specific for Escherichia coli. No confirmatory or completed tests need be performed. A national United States evaluation covering all sources and processing of water was conducted. The AC test was compared to multiple tube fermentation (quantitative) and P/A (qualitative) Standard Methods procedures. Testing protocol was that of the U.S. Environmental Protection Agency's Environmental Monitoring and Support Laboratory for establishing equivalency between methods. The quantitative comparison showed that the AC test was slightly more sensitive than MTF. For two of five sites it was more precise; for the other three it was equal. For the qualitative P/A the AC and Standard Methods analysis agreed 94%. Specificity of the AC method was established by subculturing a species of total coliform or E. coli from positives. The AC test was easy to use, can be performed by large and small utilities, and is less expensive than current methods. It can simultaneously, specifically enumerate total coliforms and E. coli without the need for confirmatory tests.