BACKGROUND: Since 1997 in which first survey for mycobacterial practices of hospitals in Korea were carried on, changes of practice and concept in mycobacterial testing have been expected because advanced testing methods have been surged for last five-year-period. We purposed to follow-up survey to monitor practices changes, and in addition, situation of quality control. METHODS: Questionnaires was composed of items including mycobacterial test methods, test volume, turnaround time (TAT), and quality control measure. It was sent to 90 laboratories of general hospitals, tuberculosis specialty hospitals and commercial laboratories in April 2001. RESULTS: Sixty-seven (74%) of 90 laboratories replied to this survey. Five of 67 laboratories (7%) were using fluorochrome method for AFB stains. In five among 58 laboratories that performed AFB cultures (8%), liquid media have been used. Mycobacterial species was identified by molecular biologic methods or high-performance liquid chromatography in 18 laboratories (34%). Average TAT of culture and identification for Mycobacterium tuberculosis was 11 days at the laboratory using Mycobacteria Growth Indicator Tube (MGIT) 9600 system and PCR method, while that at the laboratory using Ogawa media and biochemical method was 35.8 days. TATs of susceptibility tests undertaken at three laboratories were 28-60 days. Only six laboratories were joining external quality control program, even though most laboratories wanted to participate in. CONCLUSIONS: TATs for mycobacterial culture and susceptibility tests were too long to have the laboratories take a pivotal role to control tuberculosis. To improve Korean mycobacterial laboratories, it is necessary that the national health insurance system supports the newer rapid methods for mycobacterial tests and nationwide external quality control program is introduced.