Vancomycin therapy failure due to the emergence of tolerance in pneumococci is increasing. The molecular mechanism of tolerance is not clear, but lytA and pep27 are known to be involved. Our aim was to evaluate the expression of both genes in vancomycin-tolerant Streptococcus pneumoniae (VTSP) strains. Eleven VTSP strains from a total of 309 clinical isolates of S. pneumoniae from 1997 to 2006 were classified according to the criteria of Liu and Tomasz. All VTSP strains were evaluated for susceptibility according to CLSI criteria, serotype by the Quellung test, and clonality by PFGE. The expressions of lytA and pep27 were analyzed in different growth phases by RT-PCR with and without vancomycin. Eighty-two percent of VTSP strains showed resistance to penicillin, and 100% were sensitive to vancomycin and cefotaxime. The most frequent serotypes of VTSP strains were 23F (4/11) and 6B (3/11). Clonal relationship was observed in only two strains. No significant changes were observed in pep27 expression in the three phases of growth in VTSP strains with and without vancomycin. Interestingly, pep27 expression in the stationary phase in the non-tolerant reference strain R6 was significantly higher. However, no significant differences in lytA expression were observed between VTSP and R6 strains during the phases of growth analyzed. The absence of changes in pep27 expression in VTSP strains in the stationary phase may be related to their ability to tolerate high antibiotic concentrations, and thus, they survive and remain in the host under the antibiotic selective pressure reflected in therapeutic failure.