The genetic background for β-lactamase-mediated resistance to β-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla _TEM-1, of which bla _TEM-1b was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla _TEM-1a (eight E. coli, one Salmonella) and bla _TEM-1c (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE β-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC β-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla _TEM genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum β-lactamases were detected. Two new variants of bla _TEM were detected, which have been designated bla _TEM-127 and bla _TEM-128. In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum β-lactams.