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The present study was set up to determine vancomycin tolerance in Colombian, multiresistant, invasive, paediatric isolates of Streptococcus pneumoniae. A total of seven clinical isolates of penicillin-resistant S. pneumoniae, serotype 14, corresponding to the Spain9v-3 clone, were studied. Isolates were grown in Todd Hewitt broth at 37°C. Growth (A595) and cell viability (cfu/mL) were monitored hourly for 4 h after addition of vancomycin at 10× MIC. S. pneumoniae R6 was used as a control for growth. For R6, the mean decrease in A595 and cell viability (log10 cfu/mL) after 4 h of vancomycin treatment (in 10 independent experiments) was 95.1% and 4.2 log10, respectively. Six out of seven isolates exhibited similar behaviour. In contrast, for S. pneumoniae E239, the mean decrease in A595 and cell viability was 48.5% and 2.2 log10 after 4 h of vancomycin exposure, which suggested the presence of tolerance to vancomycin. The emergence of vancomycin-tolerant strains is of great concern in Colombia, owing to the alarming increase in resistance to penicillin.