Vaccination of rats against nicotine reduces nicotine distribution to brain even at nicotine doses greatly exceeding the estimated binding capacity of the available antibody. This observation suggests a differential effect by which vaccination reduces nicotine distribution to brain to a greater extent than to other tissues. To test this hypothesis, vaccinated rats received a single intravenous nicotine dose equal to twice the estimated binding capacity of nicotine-specific antibody in vaccinated rats. The total and bound serum nicotine concentrations were higher in the vaccinated rats compared to controls, while the unbound serum nicotine concentration was lower. Distribution of nicotine to brain was reduced in vaccinated rats in a time-dependent manner, with a greater reduction at 1 min (64%) than at 25 min (45%). Vaccination reduced nicotine distribution to muscle, testis, spleen, liver, heart, and kidney, but to a lesser extent than to brain, while nicotine distribution to fat was increased. Chronically infused nicotine showed a similarly altered pattern of tissue distribution in vaccinated rats, but differences were in general smaller than after a single nicotine dose; brain nicotine concentration was 24% lower in vaccinated rats, while lung nicotine concentration was higher. The presence of nicotine-specific antibody in tissues may have contributed to the increased nicotine concentrations in fat and lung. These data suggest that vaccination reduces nicotine distribution to brain not only by sequestering nicotine in serum but also by redirecting tissue distribution disproportionately away from brain, such that nicotine concentrations are reduced to a greater extent in brain than in other tissues.