Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen, and ruminants are recognized as the main natural reservoir. The purposes of this study were to detect E. coli O157 in bovine feces and surface water in a beef cattle farm of Gualeguaychú, Argentina; to characterize the isolates; and to establish the clonal relatedness by pulsed-field gel electrophoresis. Between September 2005 and November 2006, 288 samples of bovine feces and 79 samples of water troughs were studied. E. coli O157 was detected by immunomagnetic separation and polymerase chain reaction as screening techniques. The rfb(O157) gene was detected in 3.8% of the 288 fecal samples and in 17.7% of the 79 water samples. The stx gene was detected in all rfb(O157)-positive fecal samples and in 5.1% of water samples. Eleven E. coli O157 strains isolated from bovine fecal samples and eight from water samples were characterized. The most frequent stx genotype identified was stx(1) and stx(2c(vh-a)). Twelve (63.2%) strains harbored fliC(H7), eae, and ehxA genes. Using pulsed-field gel electrophoresis with the enzyme XbaI, a total of eight patterns with at least 72.1% similarity were identified among the 19 strains. The patterns of 15 strains were grouped into four clusters: two of them included only bovine strains and the other two only aquatic strains. No genetic correlation was established between the bovine and water STEC strains detected. The prevalence of STEC O157:H7 established in the herd studied was higher than that previously reported for Argentine grazed cattle.