A clinical, microbiological and economic study of a national rapid molecular service for the identification of Mycobacterium tuberculosis and the determination of rifampicin resistance in smear-positive sputum samples (and other primary specimens) was performed. Ninety-one primary specimens, of which 55 were smear-positive sputum, were examined by molecular and conventional assays. Concordance of molecular results from smear-positive sputum specimens with tuberculosis diagnosis and rifampicin resistance by conventional analysis was 52 (94.5%) of 55 and 44 (91.7%) of 48, respectively. Concordance of molecular analysis on all primary specimens was 81 (89.0%) of 91 (diagnosis) and 55 (90.2%) of 61 (rifampicin resistance). Approximately 28 days were saved in the time to diagnosis by using the molecular assay. Hospitals can reduce the cost of inappropriate isolation of patients with risk factors for multiple drug-resistant tuberculosis (MDRTB) who subsequently are shown to have drug-sensitive tuberculosis. At one hospital potential annual savings were between pound sterling 50000 and pound sterling 150000. Of the nine MDRTB cases identified, all had a previous diagnosis of tuberculosis, 78% were born overseas, 44% were known to be non-compliant with therapy, but only one case (12.5%) was HIV positive. HIV status was not significantly different between MDRTB and drug-sensitive tuberculosis cases. Over 75% of specimens were taken while the patient was on therapy. Isolates from >50% of the MDRTB cases were resistant to three or more drugs and one was resistant to seven drugs. All patients were placed on additional therapy once the molecular result was known; this was subsequently modified based on the results of in-vitro drug susceptibility testing. All survived at least 6 months of follow-up. There was no difference in the proportion of successful cultures from smear-positive samples from patients with drug-sensitive tuberculosis or MDRTB who were on therapy. Molecular rifampicin resistance assays are reliable for diagnosis in cases with smear-positive disease.