Escherichia coli is a routinely used microbiological indicator of water quality. To determine whether holding time and storage conditions had an effect on E. coli densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods. During all three phases of the study, E. coli samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection. During phase 1, when 4°C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl--D-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study. During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG). When evaluated by the Colilert method, five of seven sites showed no significant difference in E. coli density between time 0 and 48 h. During phase 3, 8 of 13 sites showed no significant differences in E. coli densities between time 0 and the 48-h holding time, regardless of method. Based on the results of these studies, it appears that if samples are held below 10°C and are not allowed to freeze, most surface water E. coli samples analyzed by commonly used methods beyond 8 h after sample collection can generate E. coli data comparable to those generated within 8 h of sample collection. Notwithstanding this conclusion, E. coli samples collected from surface waters should always be analyzed as soon as possible.Escherichia coli testing is an important tool used by public health experts for the prevention of waterborne disease. The detection of E. coli in a water sample from an environmental source provides direct evidence of fecal contamination. Regulatory agencies are increasingly requiring more emphasis on E. coli testing as part of programs aimed at curtailing waterborne disease. Holding time and temperature can have a significant impact on the density of microbiological indicators at the time of sample analysis (4, 5, 7). Recommendations for E. coli holding times range from 8 h (2, 3, 9) to 24 h (8), and holding temperatures below 10°C are generally considered acceptable (2,3,8,9). It is also recommended that when transport conditions result in delays longer than 6 h, the use of field laboratory facilities located at the site of collection or delayed incubation procedures be considered (2). The Surface Water Treatment Rule requirements of the U.S. Environmental Protection Agency (USEPA) for total coliform and fecal coliform monitoring of surface water used as drinking water sources (3) specify that the time from sample collection to initiation of analysis is not to exceed 8 h; the regulations also encourage (but do not require) drinking water system personnel to hold samples at below 10°C during transit.Unfortunately, data from evaluations of microbiological indicator density that support current holding time recommendations are limited, particularly for E. coli....