A total of 184 Escherichia coli isolates recovered from cows with acute mastitis were examined for recognized pathogenic mechanisms and virulence factors commonly found in pathogenic groups of E coli. A modification of the Eng procedure (for detecting complement deficiencies in serum) was used to test for resistance to different animal sera. The Sereny test (for invasiveness), infant mouse test (for heat-stable enterotoxin), and Y-1 adrenal tumor cell assay (for heat-labile enterotoxin) were used. Hemagglutination tests, using rabbit, sheep, and guinea pig RBC, were done with and without added mannose. All of the 184 isolates were serum resistant in all tested sera. None of the isolates was invasive. Only 1 isolate was positive for heat-stable enterotoxin and 2 cultures were positive for heat-labile enterotoxin. Multiple patterns of hemagglutination were observed. The majority of the isolates exhibited both mannose-sensitive and mannose-resistant hemagglutinins with guinea pig and rabbit RBC. A few strains were positive only in mannose-sensitive or mannose-resistant hemagglutination tests. A few strains were negative in all hemagglutination tests. Based on our results, E. coli from cows with acute mastitis lack the virulence factors commonly observed in other E coli groups associated with disease. Serum resistance was the only characteristic that could be related to virulence.