E. coli are fecal coliform bacteria that are frequently used as indicators of water fecal contamination.  An 8-hour sample holding time is generally recommended for E. coli surface samples, but it is not always feasible to meet holding time requirements when sampling remote environments.  Several studies have evaluated the effects of extending holding times, but the results were contradictory. We investigated how extended holding times affect E. coli densities with samples collected at sites with known low (LDS), medium (MDS), and high (HDS) ambient E. coli densities. All samples were stored on ice and processed with IDEXX system at 6, 8, 10, 12, 18, and 24 hours after collection. Density geometric means decreased with increasing holding times at all sites. The coefficient of variability (CV) was lowest at the HDS and highest at the LDS. Overall, there were no significant effects of holding times at the MDS. However, there were significant differences between 8h and 24h holding times at the HDS and between 8h and 6h & 18h holding times at the LDS. These results suggest that sample holding times can potentially be extended with the exception of low-density sites, which are less of a concern to human health.