Structurally distinct nicotine immunogens can elicit independent antibody responses against nicotine when administered concurrently. Co-administering different nicotine immunogens together as a multivalent vaccine could be a useful way to generate higher antibody levels than with monovalent vaccines alone. The immunogenicity and additivity of monovalent and bivalent nicotine vaccines was studied across a range of immunogen doses, adjuvants, and routes to assess the generality of this approach. Rats were vaccinated with total immunogen doses of 12.5 - 100 μg of 3'aminomethyl nicotine conjugated to recombinant Pseudomonas exoprotein A (3′-AmNic-rEPA), 6- carboxymethylureido nicotine conjugated to keyhole limpet hemocyanin (6-CMUNic-KLH), or both. Vaccines were administered s.c. in alum or i.p. in Freund's adjuvant at matched total immunogen doses. When administered s.c. in alum, the contributions of the individual immunogens to total nicotine-specific antibody (NicAb) titers and concentrations were preserved across a range of doses. Antibody affinity for nicotine varied greatly among individuals but was similar for monovalent and bivalent vaccines. However when administered i.p. in Freund's adjuvant the contributions of the individual immunogens to total NicAb titers and concentrations were compromised at some doses. These results support the possibility of co-administering structurally distinct nicotine immunogens to achieve a more robust immune response than can be obtained with monovalent immunogens alone. Choice of adjuvant was important for the preservation of immunogen component activity.