Technical Abstract: A new temperature-conditional shuttle vector, pBB80C, was constructed and utilized to generate an in-frame deletion in the leukotoxin structural gene of Mannheimia haemolytica serotype 1. Culture supernatants from the mutant contained no detectable cytotoxicity to BL-3 lymphocyte targets, and contained a new protein of approximately 66 kDal MW which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the 100-105 kDal MW of native LktA. Calves vaccinated mucosally by top-dressing the live mutant onto feed, or parenterally by subcutaneous injection, were resistant to virulent challenge with the parent strain. Serologic antibody response, reduction in lung lesion, and reduction in pulmonary infectious load was greater among calves mucosally vaccinated than those which were vaccinated by injection.