From December 1998 through February 1999, a study was conducted in a Brucella-infected bison herd to evaluate the safety of booster vaccination of adult bison (Bison bison) with 6×10⁹ colony forming units (CFU) of Brucella abortus strain RB51 (SRB51) that had previously been vaccinated as yearlings with 1×10¹⁰ CFU of SRB51. Abortions or other adverse effects were not observed after SRB51 booster vaccination. At 10 wk after adult vaccination, pregnant and nonpregnant bison (n=65) were randomly selected for bacteriologic sampling of targeted maternal tissues during abattoir processing. Fetal tissues were also sampled in pregnant bison. The SRB51 vaccine was recovered from tissue samples of eight of 48 pregnant bison and none of 17 nonpregnant bison. In three of the eight culture-positive bison, SRB51 was recovered from fetal tissues. In three additional bison, one pregnant and two nonpregnant, B. abortus biovar 1 field strain was recovered from internal iliac or supramammary lymphatic tissues. Results of this study suggest the possibility that the SRB51 vaccine can be safely used to booster vaccinate pregnant bison in a Brucella-infected bison herd. Our data also reaffirms the potential for B. abortus field strains to persist in bison until attainment of reproductive age, despite extensive use of vaccination and serologic testing.