ABSTRACT: BACKGROUND: Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control--5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle. RESULTS: Overall, patterns of antimicrobial resistance expressed by E. coli isolates did not change among diet groups (CON vs. antibiotic treatments), however; isolates obtained on selective plates (i.e., M(A),M(T)), exhibited multi-resistance to sulfamethoxazole and chloramphenicol more frequently when obtained from TS-fed steers than from other treatments. Antibiograms and PFGE patterns suggested that AMR E. coli were readily transferred among steers within pens. Most M(T) isolates possessed the tet(B) efflux gene (58.2, 53.5, 40.8, and 50.6% of isolates from CON, T, TS, and V steers, respectively) whereas among the M(A) (ampicillin-resistant) isolates, the tem1-like determinant was predominant (occurring in 50, 66.7, 80.3, and 100% of isolates from CON, T, TS, and V steers, respectively). CONCLUSIONS: Factors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR E. coli among feedlot cattle.