The characteristics of laboratories performing tuberculosis (TB) diagnostic procedures were investigated in ten Italian Regions, through a mailed questionnaire. Three hundred and eighty laboratories answered (70.8 % response rate), 250 of which performed directly at least one TB diagnostic procedure. Standard criteria concerning microscopy, culture, identification, and drug susceptibility testing were frequently not satisfied, particularly those related to the volume of activity (32 % of laboratories performing microscopy examined 10 samples and 36 % of those performing culture performed 20 cultures per week), processing time, biosafety requirements and participation to internal/external quality control programs. The survey' results highlight the need to promote the adoption of standardized procedures and to centralize the mycobacteriology testing in a reduced number of high quality laboratories, in order to improve diagnostic accuracy, resource management and quality of surveillance data.