All non-private hospitals in Malawi that registered TB cases in 2001, during which there was a bus service for transporting sputum specimens to the Central Reference Laboratory (CRL) for mycobacterial culture and drug sensitivity testing (CDST). To determine the performance of the system of collecting and processing sputum specimens from patients with recurrent smear-positive pulmonary TB through to CDST. Structured interviews with TB Officers, and retrospective data collection using TB and laboratory registers. There were 964 patients with recurrent smear-positive PTB. TB Officers took responsibility for collecting and transporting sputum to the CRL, and 73% reported using the bus service. Sputum specimens from 384 (40%) patients arrived at the CRL. Of these, 40% were found to have negative concentrated smears at the CRL, and 36% of specimen sets arriving at CRL were successfully cultured for DST. Most specimens had been collected after the start of anti-tuberculosis treatment. Although delays in collection adversely affected culture, only 43% of specimen sets collected on or before the first day of treatment yielded Mycobacterium tuberculosis. Problems were identified at all stages of the system and strategies to remedy these are being put in place.