The occurrence of the enterococcal surface protein (esp) gene in the opportunistic pathogens Enterococcus faecalis and E. faecium is well-documented in clinical research. Recently, the esp gene has been proposed as a marker of human pollution in environmental waters; however, information on its relative incidence in various human and animal fecal sources is limited. We have determined the occurrence of the esp gene in enterococci from human (n = 64) and animal (n = 233) fecal samples by polymerase chain reaction using two primer sets: one presumably specific for E. faecium (esp_fm) and the other for both E. faecalis and E. faecium (esp_fs/fm). We believe that this research is the first to explore the use of esp_fs/fm for the detection of human waste in natural environmental settings. The incidence in human sources was 93.1% esp_fm and 100% esp_fs/fm in raw sewage influent; 30% for both esp_fm and esp_fs/fm in septic waste; and 0% esp_fm and 80% esp_fs/fm in active pit toilets. The overall occurrence of the gene in animal feces was 7.7% (esp_fs/fm) and 4.7% (esp_fm); animal types with positive results included dogs (9/43, all esp_fm), gulls (10/34, esp_fs/fm; 2/34, esp_fm), mice (3/22, all esp_fs/fm), and songbirds (5/55, all esp_fs/fm). The esp gene was not detected in cat (0/34), deer (0/4), goose (0/18), or raccoon (0/23) feces. The inconsistent occurrence, especially in septic and pit toilet sewage, suggests a low statistical power of discrimination between animal and human sources, which means a large number of replicates should be collected. Both esp_fm and esp_fs/fm were common in raw sewage, but neither one efficiently differentiated between animal and other human sources.