This study was conducted to evaluate canine parvovirus disease prevention efficacy of the minimum immunizing dose of the CPV-2b fraction of a multivalent vaccine when administered at approximately 6 weeks of age to pups with maternal CPV-2b antibodies. A second dose was administered 4 weeks later. Pups were challenged with a virulent strain of CPV-2c virus 2 months after the second vaccination. Efficacy was evaluated by monitoring the pups for various clinical observations and laboratory testing of parvovirus infection, including mucous stool, bloody stool, diarrhea, fever, death, leukopenia, lymphopenia, CPV-2b serum neutralization titer, and detection of CPV in the feces. Upon a severe challenge with a virulent CPV-2c virus, four of five (80%) control pups had at least three of four clinical signs of CPV infection while 19 of 20 (95%) vaccinated pups had not more than one sign of CPV infection. The response of the control pups confirmed the virulence of the challenge and validity of the study. The response of the vaccinated pups demonstrated the efficacy of the CPV-2b vaccine, even in puppies with maternal antibody, which was one of the main objectives of this study. The outcome of this study was consistent with the 9CFR requirements necessary to support an additional label claim that the vaccine aids in the prevention of disease caused by CPV-2c when administered to puppies as young as 6 weeks of age with maternal CPV antibodies.