Oligonucleotide primers were used in polymerase chain reaction assays to detect tetracycline-resistant determinants Tet K and Tet L. Forty-three isolates representing 11 genera carrying Tet K and/or Tet L determinants gave appropriate PCR products. The PCR products hybridized with labelled Tet K or Tet L probes, and differentiated the Tet K from the Tet L PCR products. We confirmed that two Haemophilus aphrophilus carried the Tet K determinant and two Veillonella parvula carried the Tet L determinant. This is the first time that either of these two genes have been found in Gram-negative species. Four vaginal samples, previously positive with the Tet M/O PCR assay, were also positive with the Tet K/L assay. This is the first report of a PCR assay for the detection of Tet K and Tet L determinants in bacteria or clinical specimens.