The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17. ؊1 in seawater diluted 1:10 with freshwater to Ϸ5,000 CFU ml ؊1 in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU·100 ml ؊1 , while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU·100 ml ؊1 . The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.Coliform bacteria are widely used as indicators of fecal contamination of both fresh and marine waters. Certain members of the coliform group live outside of the gastrointestinal tract in the environment and may create a false indication of fecal contamination. A more specific indicator of fecal contamination is Escherichia coli, a thermotolerant fecal coliform bacterium distinguished by its ability to grow at 44.5°C and by its expression of the enzyme ␤-D-glucuronidase. This enzyme hydrolyzes 4-methylumbelliferyl-␤-D-glucuronide (MUG) to form a fluorescent (F) product that is visible with UV light (11,23) and that is used to differentiate E. coli from other fecal coliforms.The Colilert-18 defined-substrate technology system (Idexx Laboratories, Inc., Westbrook, Maine) is intended to provide rapid (18-h), standardized quantitation of total coliforms and E. coli. Various versions of the Colilert system were shown to yield results that are statistically consistent with the standard methods of membrane filtration and multiple-tube fermentation testing used for the detection of coliforms and E. coli in freshwater (2, 3, 6, 7). The U.S. Environmental Protection Agency has approved Colilert for use in drinking water monitoring (9, 10), and analyses of E. coli in fresh (6, 12, 21) and saline (18, 22) natural waters have been published.The Colilert-18 assay system is based on a defined substrate medium containing MUG and ortho-nitrophenyl-␤-D-galactopyranoside (ONPG) and is used for the one-step detection of both total coliforms and E. co...