The C-terminal region of the pbp5 gene was sequenced in 11 ampicillin-resistant and 5 ampicillin-susceptible Enterococcus faecium isolates of animal origin, and compared with a pbp5 reference sequence (GenBank accession no. X84860). Eight different pbp5 alleles (designated A–H) were detected when amino acid changes in the region 461–629 were considered. Three of these alleles (A–C) were detected in ampicillin-susceptible isolates (MIC range 1–8 μg ml−1), and included the changes 470H→Q, 471V→I, 487Q→L, 581I→V, 595E→A or 622E→D. The remaining five alleles (D–H) were found in ampicillin-resistant isolates (MIC range 32–256 μg ml−1); three of these alleles (F–H) presented a serine insertion at position 466′, in addition to other important amino acid changes (485M→A, 496N→K, 499A→T, 525E→D, 586V→L or 629E→V). The other two alleles presented the amino acid changes 496N→K and 629E→V (allele D), and 470H→Q (allele F). A correlation between deduced amino acid changes in PBP5 and ampicillin MICs was detected in animal E. faecium isolates.