Vancomycin, a glycopeptide antimicrobial agent, represents the last line of defence against a wide range of multi-resistant Gram-positive pathogens such as enterococci, staphylococci and streptococci. However, vancomycin-resistant enterococci and staphylococci, along with vancomycin-tolerant clinical isolates, are compromising the therapeutic efficacy of vancomycin. It is conceivable that tolerance may emerge during prolonged vancomycin use. It has not been until recently, however, that the molecular basis of this tolerance began to be understood. Superoxide anions might be involved in the bactericidal activity of vancomycin in enterococci, and recent evidence suggests that the stringent response is partly responsible for vancomycin tolerance inEnterococcus faecalis. The mechanism of vancomycin tolerance inStaphylococcus aureusandStreptococcus pneumoniaeis sometimes associated with a reduction of autolysin activity. Vancomycin tolerance inS. aureusandS. pneumoniaealso appears to be somehow related with the two-component regulatory systems linked to cell envelope stress, although the precise molecular regulatory pathways remain poorly defined.