Paenibacillus larvae is the causal agent of American Foulbrood (AFB) disease, the most virulent bacterial disease of honeybee (Apis mellifera L.) brood. Oxytetracycline is the main antibiotic used for prevention and control of AFB. Using the polymerase chain reaction, isolates were screened for the presence of the tetracycline resistance tet(K) and tet(L) determinants. Four isolates (5%), which correlated with the Tc-resistant phenotypes, were found to carry the tet(K) determinant, whereas none carried the tet(L) determinant. P. larvae cells were also screened for the presence of extrachromosomal DNA and evidence obtained that tetracycline resistance is plasmid-encoded. A few P. larvae isolates were found to be able to transfer the tet(K) determinant to Bacillus subtilis, suggesting that a conjugation mechanism may be involved in the transfer of the tetracycline-resistant phenotype. Minimum inhibitory concentrations to tetracycline were determined for 75 isolates of P. larvae from different geographical origins and found to range between 0.062 and 128 microg tetracyclineml(-1), with MIC(50) and MIC(90) values of 1 and 4, respectively. According to results from P. larvae populations, isolates could be considered as susceptible when their MICs were <4, intermediate for MICs values 4-8 and resistant for MICs > or = 16. To our knowledge, this is the first report of Tc(r)Paenibacillus species carrying a tet(K) gene, and also the first record of P. larvae strains carrying tet(K) determinants and its correlation with the presence of extrachromosomal DNA.