Biochemical and serological characterizations were determined on 184 strains of Escherichia coli isolated from acute cases of bovine mastitis. The majority of the test reactions were in agreement with the results reported by others for E coli. In those characteristics reported as variable, no trends or patterns unique to the bovine strains were observed, except in the adonitol fermentation. Approximately half of the cultures were positive. Growth on T-7 agar plates exhibited three types of colony morphology. Rough and intermediate textures predominated over those with smooth colony surfaces. A large variety of O and K serogroups was detected and identified, but many K antigens showed multiple low titer agglutinations and therefore were untypable with available antisera. The results did not provide markers to distinguish mastitis strains of E coli from those found in other diseases or environments;Several pathogenic and virulence mechanisms commonly associated with E coli isolated from disease processes other than mastitis were studied. Only one strain was found to produce heat-stable toxin in the infant mouse test and two strains were positive for the heat-labile toxin production in the Y-1 adrenal tumor cell bioassay. None of the cultures were found to be invasive in the Sereny test and all the isolates were serum resistant. The majority of the E coli agglutinated guinea pig red blood cells in a mannose-sensitive (MS) manner suggesting the presence of type 1 pilus and rabbit red blood cells in a mannose-resistant way, indicating another type of adhesin. Based on our results, it appears that serum resistance is the only characteristic common to all isolates that could possibly be related to virulence.