Polyclonal antibodies are typically produced in rabbits. The rabbit's health plays an important role in the quality of antibodies produced. Therefore, recommendations have been made by organizations on which bacteria and how frequently to test rabbit colonies. Since it is well known that rabbits may contain cross-reactive antibodies in their preimmune serum, it is common to test the rabbits for reactivity prior to immunization. Here preimmune sera from 19 different rabbits were tested with ELISA against 27 pathogenic and nonpathogenic bacterial cultures. ELISA results showed that Salmonella enterica serovar Typhimurium and Bacillus cereus AS4-12 had the highest average absorbance values (0.60 and 0.54, respectively) and the most preimmune serum samples testing positive was 17. Pseudomonas putrefaciens and B. subtilis had the lowest absorbance values (< 0.1) and did not test positive in any of the preimmune serum samples. Fourteen of the 27 cultures showed positive reactions with 50% or more of the preimmune serum samples tested. Fifty-three percent of the rabbit preimmune sera showed positive reactions with 10 or more bacterial cultures. In Western blot analyses, selected serum samples showing the highest ELISA values reacted with bands in the 97, 36, and 29 kDa regions or with bands in the 63 kDa and 32 kDa regions. Data suggest that the presence of cross-reactive antibodies in the preimmune serum is a common problem amongst the samples tested. Extensive preimmune serum testing should be implemented when polyclonal antibodies are intended for diagnostic testing.