A clinical strain of Enterococcus faecium ATCC 51559 exhibits heteroresistance, i.e. a high level of resistance to vancomycin (minimum inhibitory concentration (MIC)>256 microg/mL) by broth dilution but sensitivity to vancomycin by Etest (MIC=1.8 microg/mL). Three variants of this strain, EF1, EF2 and EF3, exhibit high levels of resistance to vancomycin both by broth dilution and Etest assays. The four strains were used to study heteroresistance by pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequence analysis of a partial region of the van operon. Minor differences between SalI and SmaI restriction profiles of the variants and the parental strain were observed by PFGE analysis. PCR analysis confirmed the presence of the vancomycin resistance marker vanA (0.73 kb) and a larger than expected amplicon (8.2 kb vs. 6.7 kb) of the van operon in all the strains. The 8.2kb van operon was cloned for EcoRI RFLP and sequence analysis. All of the clones exhibited distinctly different RFLP profiles when grown in the presence of kanamycin or vancomycin+kanamycin. The presence of these antibiotics during overnight growth of EF1 on plates also resulted in altered SalI PFGE profiles. Sequence analysis of the van operon clones revealed a 1.5kb IS1251-like insertion element between the vanS and vanH genes in all the strains. Several novel point mutations in the vanR, vanS, vanH, vanA, vanX and vanY genes were also discovered. Some of these mutations were present in the parental strain only and included base substitutions T-->C, A-->G, T-->A and T-->C at nucleotide positions 4202, 4597, 4763 and 6207 of Tn1546, resulting in amino acid replacements I76-->T and K208-->E of vanR, S19-->T of vanS and L64-->P of vanH genes, respectively. We believe that these are responsible for the observed heteroresistance. The present study clearly shows how independent novel mutations can give rise to polymorphism, heteroresistance and clonal diversity among vancomycin-resistant enterococci strains as a result of continuous exposure to antibiotics.