The present studies were carried out in order to investigate the cross-reaction of botulinum neurotoxins (BoNTs) with human and mouse antibodies against tetanus neurotoxin (TeNT) and determine whether injection of BoNT into a host that has been primed with TeNT would result in boosting of the response to the injected BoNT. Human antisera against TeNT obtained from 9 individuals were found to exhibit substantial cross-reaction with BoNTs A and B. We prepared antibodies (Abs) against inactivated tetanus neurotoxin (TeNT) in outbred mice and determined the binding of these Abs to active TeNT and active botulinum neurotoxins (BoNTs) A and B. Blood samples were collected before immunization (day 0) and on days 42, 82 and 125 after the first injection. The reactions of these sera with the immunizing antigen (inactivated TeNT), active TeNT, active BoNT/A and active BoNT/B were determined. At a fixed dilution (1:62.5 v/v), the sera contained high levels of Abs that reacted with TeNT and also with BoNTs A and B. Throughout the test period (up through day 125) and at different dilutions the cross-reactions of the antisera with BoNT/B were almost twice those with BoNT/A. The reactions of the antisera with the immunizing antigen (inactive TeNT) or with active TeNT were essentially equal throughout the dilution range tested (1:16-1:500 v/v). To determine whether injection of BoNT/A or B into a host that had been primed with TeNT resulted in boosting of the response to the priming antigen (TeNT) as well as BoNT/A or B, mice were primed with TeNT and boosted 21 days later with TeNT, BoNT/A or BoNT/B. Appropriate controls were also employed. Blood samples were collected prior to TeNT priming (day -1) and on days 21, 32, 46 and 67 after priming. In TeNT-primed mice, BoNTs A or B boosted the anti-TeNT Ab responses slightly but had no significant boosting effect on the Ab populations that bind to BoNTs A or B. It is concluded that while Abs against TeNT cross react with BoNTs and the cross reaction with BoNT/B is almost double that of BoNT/A, injection of BoNTs A or B in the presence of a prior active immunity against TeNT is not very likely to make the host mount an Ab response against the injected BoNT.