Antibiotics are essential and widely used, both for therapeutic and non-therapeutic purposes. Careless and excessive use of antibiotics, however, is compromising their efficacy by selecting for resistant bacteria. In this study, we investigated the role of manure from animal farms using antibiotics for growth-promotion and prophylaxis. Soils were sampled from fields fertilized with manure from 3 dairies (using antibiotics solely for veterinary purposes), 3 large concentrated animal feeding operations (using subtherapeutic antibiotics) and 3 non-agricultural areas. No significant differences were detected in resistance to chlortetracycline, tylosin or carbadox among all these sites. Chlortetracycline resistant isolates detected were similar, and were predominantly related to Streptomyces, Variovorax and Chryseobacteria spp. Tetracycline resistance conferring genes were rarely detected in the resistant isolates. However, soils collected from a small animal farm using subtherapeutic chlortetracycline differed significantly from all these sites. At this farm, waste was not applied to soil at agronomic rates, but instead it was allowed to accumulate outside the animal pen. This site exhibited a 10-100 fold increase in resistance to chlortetracycline in the surrounding soil. The resistant bacteria isolated were markedly different from the other sites and were primarily Bacillus spp. and Pseudomonas spp.. A significant fraction of these isolates also harbored one or more genes conferring tetracycline resistance. In follow-up studies at this farm, we observed no reduction in resistance levels nearly 2 years after the farm stopped its animal feeding operation.