The incidence of antibiotic resistance was compared in Gram-negative bacteria isolated from the intestinal tracts of catfish and from water and sediment in aquaculture ponds and rivers of the south-eastern United States. Resistance to tetracycline, oxytetracycline, chloramphenicol, kanamycin, ampicillin, and nitrofurantoin was determined. The predominating microflora were Plesiomonas shigelloides and Aeromonas hydrophila. Individual and multiple antibiotic resistances were associated with antimicrobial use. Resistance apparently was higher in ponds undergoing antimicrobial therapy or with a history of recent treatment than in ponds without recent antimicrobial treatment. The lowest incidence of resistance was found in riverine bacteria.