Many water utilities are required to monitor source water for the presence of total coliforms, fecal coliforms, or both. The Colilert system, an application of the defined substrate technology, simultaneously detects the presence of both total coliforms and Escherichia coli directly from a water sample. After incubation, the formula becomes yellow if total coliforms are present and fluorescent at 366 nm if E. coli is in the same sample. No confirmatory tests are required. The Colilert system was previously assessed with distribution water in a national evaluation in both most-probably-number and presence-absence formats and found to produce data equivalent to those obtained by using Standard Methods for the Examination of Water and Wastewater (Standard Methods). The Colilert system was now compared with Standard Methods multiple-tube fermentation (MTF) for the enumeration of total coliforms and E. coli from surface water. All MTF tubes were confirmed according to Standard Methods, and subcultures were made to identify isolates to the species level. The Colilert system was found equally sensitive to MTF testing by regression, t test, chi-square, and likelihood fraction analyses. Specificity of the Colilert system was shown by the isolation of a species of total coliform or E. coli after the appropriate color change. The Colilert test can be used for source water samples when enumeration is required, and the benefits previously described for distribution water testing--sensitivity, specificity, less labor, lower cost, faster results, no noncoliform heterotroph interference--are applicable to this type of water analysis.