Is a multiple number of sputum specimens necessary for the diagnosis of pulmonary tuberculosis? To answer this question, 6844 respiratory specimens obtained from previously untreated patients suspected of having pulmonary tuberculosis between 1998 and 2001 were evaluated retrospectively. All of the specimens were evaluated by acid fast bacilli smear and BACTEC 460 TB culture system. A total of 785 (11%) specimens from 353 patients were positive for Mycobacterium tuberculosis complex. For 76% (270/353) of these patients the organism was detected from sputum specimens collected sequentially for daily basis. Mycobacterium tuberculosis was isolated in the first, second and third samples of the majority (98%,195/199) of patients who had three or more sputum samples sent to the laboratory. Our results indicate that, we could carry out Mycobacterium tuberculosis isolation in the first, second and third sputum samples of the overwhelming majority of the patients and the diagnostic value of four or more sputum specimens submitted to the laboratory was very low (2%). We recommend that, for definitive and cost-effective diagnosis of pulmonary tuberculosis at least three sequential sputum specimens be collected for all patients suspected pulmonary tuberculosis.