Efficacy of an Escherichia coli (O111:B4) J5 bacterin for preventing naturally occurring IMI and clinical mastitis was tested in a 2.5-yr field trial in a 225-cow commercial herd. Cows with odd-numbered identification were vaccinated, and cows with even-numbered identification served as unvaccinated controls for each lactation during the study. Immunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula at drying off, 30 d after drying off, and at calving. Percentage of quarters infected at calving with Gram-negative bacteria did not differ between treatment groups. A total of 67% of Gram-negative bacterial IMI present at calving in control cows became clinical during the first 90 d of lactation compared with 20% in vaccinated cows. Rate of Gram-negative bacterial clinical mastitis was higher in control cows than in vaccinated cows during the first 90 d of lactation. Immunization with the E. coli J5 bacterin did not reduce level of Gram-negative bacterial IMI at calving but did reduce incidence of clinical mastitis.