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Microbiological findings at diagnosis and at the end of treatment are relevant for evaluating tuberculosis (TB) treatment programmes. The objectives of this study were to describe the microbiological findings at diagnosis and at the end of treatment in pulmonary and extrapulmonary TB patients treated under programme conditions in Italy. The study was a prospective monitoring activity based on the collection of standard recording and reporting forms from a representative sample of Italian TB Units. The forms with individual data were reviewed and analysed on a quarterly basis, 9 months after enrolment. The complete bacteriological profile of patients was analysed at diagnosis and at the completion of treatment. Individual data on 992 patients were analysed. At diagnosis 320 (32.2%) of cases were pulmonary sputum smear positive, 361 (36.4%) pulmonary smear negative or not done and 311 (33.4%) extrapulmonary; 424 (42.7%) of all TB cases were culture confirmed at diagnosis (368, 50.2%, of pulmonary cases); 575 (84.4%) of pulmonary cases had a culture done at diagnosis and 156 (22.9%) at the end of treatment (p < 0.001); 572 (84%) had a sputum smear done at diagnosis and 164 (24.1%) at the end of treatment (p < 0.001). Although the rate of bacteriologically confirmed cases is similar to that of other European countries, the bacteriological confirmation at diagnosis and, particularly, at the end of treatment, is sub-optimal. The importance of further disseminating national guidelines among physicians managing TB is emphasized, in order to achieve a higher proportion of TB cases bacteriologically confirmed at diagnosis and monitored at the end of treatment.