A prospective study of the clinical efficacy of an aminoglycoside antibiotic (streptomycin, SM) for the treatment of pulmonary Mycobacterium avium complex (MAC) disease was carried out. In a multicenter trial, patients with pulmonary MAC disease received protocol-guided combined chemotherapy with or without SM. SM was given to the patients intramuscularly 15 mg/kg three times per week for the initial 3 months and three other antibiotics (rifampicin, ethambutol, and clarithromycin) were added and administered for over 24 months after the conversion of MAC strains. From April 1998 to December 2004, 160 HIV-negative patients were enrolled in this trial. Fourteen patients were found to be ineligible because they could not continue the treatment, and they were excluded from the analysis after randomization. Seventy-three patients were assigned to receive combined chemotherapy with SM (group A) and 73 were assigned to receive combined chemotherapy without SM (group B). The median durations of treatment were 27.6 months in group A and 28.4 months in group B. The difference in the backgrounds of the groups was not statistically significant. There were no differences in microbiological and radiological findings between the groups, but the sputum conversion rate for pulmonary MAC disease at the completion of treatment was significantly higher in group A than that in group B. Although, there were no significant differences in the sputum relapse rate and clinical improvement including both clinical symptoms and radiological findings, group A showed better initial microbiological response than group B. As for adverse reactions and abnormal laboratory findings, there were no significant differences between the groups. Based on the results of this double-blind randomized study, we support treatment including SM according to both the American Thoracic Society (ATS) and the Japanese Society for Tuberculosis (JST) guidelines for patients with pulmonary MAC disease without HIV infection.