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Many watersheds are designated on the 303(d) list for non-compliance with the Clean Water Act because they exceed the TMDL for fecal coliforms. Some of these watersheds exist in remote areas where the source of fecal coliforms is undoubtedly from wildlife. If it could be documented that the source of fecal coliforms is wildlife, then it may be possible to delist these watersheds because control over wildlife is difficult, if not impossible. In this study, we used ribotyping, a method that differentiates subspecies of a single bacterial species by examining their differences in DNA encoding for ribosomal RNA, to identify the host origin of fecal contamination in a forested watershed that contained penned deer but no humans or domestic animals. Because the deer were penned and fed a known diet, this also allowed us to examine the effect of diet on ribotype diversity between penned and wild deer. Escherichia coli, a fecal coliform, was the bacterium selected. A total of 298 E. coli isolates was obtained: 48 upslope of the watershed, 50 from the downslope, 100 from penned deer, and 100 from wild deer. At a 95% similarity cutoff, 19 of 48 (39.6%) and 23 of 50 (46.0%) environmental isolates were identified as deer isolates from the up- and downslope of the watershed, respectively. In the case of diet, the number of E. coli isolates per ribotype was 3.0:1 for wild deer and 6.7:1 for penned deer. This 2.2-fold difference suggests that diet affects ribotype diversity, and that a host origin database should contain more isolates from wild than from captive animals. Given a sufficiently large host origin database, the results suggest that ribotyping can identify the source of fecal contamination to such an extent that it may be possible to delist a watershed impaired only by wildlife.