Aims: The Escherichia coli burden at a Great Lakes urban beach was evaluated during the summer months to determine if sand served as a reservoir for E. coli, and if there was evidence of cell replication in situ. Field and laboratory studies investigated the effects of moisture, temperature and UV on E. coli densities in the sand. Methods and Results: Sand samples (n = 481) were collected across three distinct transects of the beach, the top, a middle streamline, and the berm, over 15 sample days. The highest levels were found in the middle streamline, which was affected by stormwater discharge from nearby outfalls and roosting gulls; daily geometric mean levels of these seven sites ranged from 6700 to 40 900 CFU per 100 g of sand. Escherichia coli levels were greatest in samples with moisture levels between 15% and 19%, and were significantly higher than 0-4 and 20-24% ranges (P < 0.05). Pre- and post-rain samples at the beach demonstrated an increase in E. coli levels nearly 100-fold within 30 min, suggesting sand washout as a major mechanism for loading of E. coli into the beach waters. Rep PCR analysis of 160 isolates obtained from eight sites demonstrated that 21% of the isolates fell into one of the six clonal patterns, suggesting that bacteria may be able to replicate and possibly colonize beach sand. Sand field plots inoculated with E. coli cells containing pGFPuv that expresses GFP (green fluorescent protein) as a marker showed an initial two- to 100-fold increase at 24 h, depending on the temperature condition. The sand appeared to provide considerable protection from UV exposure as no significant difference was seen in cell densities within the first 2-4 cm of sand between exposed and unexposed plots (P < 0.05). Conclusions: Beach sand may act as a reservoir for E. coli. Replication of cells appears to be one possible contributing factor to the persistently high levels, as indicated by both field studies and laboratory studies, and warrants further investigation. Moisture content of sand may also be a determinant of cell persistence in the sand environment. Significance and Impact of the Study: Escherichia coli is used as an indicator organism for faecal pollution at most Great Lakes coastal beaches; therefore, a better understanding of how E. coli might survive, or possibly replicate, in the environment would improve interpretation of beach monitoring results.