The intracellular β-lactamases of 100 distinct clinical isolates of Yersinia enterocolitica were examined, to investigate the effect of selected β-lactam agents on the enzymes. The enzyme inhibition profiles obtained were specific to each of the three biotypes 1A, 3 and 4. Biotype 4 strains elaborated only one β-lactamase with a p of 8.3 corresponding to the carbenicillin-hydrolysing β-lactamase enzyme A of Cornelis & Abraham. Biotype 3 strains also produced a single β-lactamase with a pI of 5.4–5.6 corresponding to enzyme B, the chromosomally mediated inducible cephalosporinase. Two β-lactamases were found in biotype 1A strains, one was identical to enzyme A and the other, although showing a slight difference in pI to enzyme B, was similar to this enzyme in other respects. The differences in the distribution of enzyme A and enzyme B in strains of Y. enterocolitica belonging to the biotypes 1A, 3 and 4 isolated in Australia, explain the different patterns of susceptibility to β-lactam antibiotics.