Nonpoint sources of pollution that contribute fecal bacteria to surface waters have proven difficult to identify. Knowledge of pollution sources could aid in restoration of the water quality, reduce the amounts of nutrients leaving watersheds, and reduce the danger of infectious disease resulting from exposure to contaminated waters. Patterns of antibiotic resistance in fecal streptococci were analyzed by discriminant and cluster analysis and used to identify sources of fecal pollution in a rural Virginia watershed. A database consisting of patterns from 7,058 fecal streptococcus isolates was first established from known human, livestock, and wildlife sources in Montgomery County, Va. Correct fecal streptococcus source identification averaged 87% for the entire database and ranged from 84% for deer isolates to 93% for human isolates. To field test the method and the database, a watershed improvement project (Page Brook) in Clarke County, Va., was initiated in 1996. Comparison of 892 known-source isolates from that watershed against the database resulted in an average correct classification rate of 88%. Combining all animal isolates increased correct classification rates to ≥95% for separations between animal and human sources. Stream samples from three collection sites were highly contaminated, and fecal streptococci from these sites were classified as being predominantly from cattle (>78% of isolates), with small proportions from waterfowl, deer, and unidentified sources (≈7% each). Based on these results, cattle access to the stream was restricted by installation of fencing and in-pasture watering stations. Fecal coliforms were reduced at the three sites by an average of 94%, from prefencing average populations of 15,900 per 100 ml to postfencing average populations of 960 per 100 ml. After fencing, <45% of fecal streptococcus isolates were classified as being from cattle. These results demonstrate that antibiotic resistance profiles in fecal streptococci can be used to reliably determine sources of fecal pollution, and water quality improvements can occur when efforts to address the identified sources are made.