Non-tuberculous mycobacteria (NTM) found frequently in tap water and environment cause important opportunistic infections in immunocompromised patients. The aim of this study was to isolate and identify non-tuberculous mycobacteria in soil, raw milk and water distribution system samples in Mersin (a province located at Mediterranean region of Turkey). A total of 101 water, 124 soil and 40 milk samples collected from the central part and suburban parts of Mersin during November 2003-May 2004 period were included in the study. Water samples were collected from 29 different water distribution systems; soil samples from different parks and gardens and milk samples from raw milks sold at different districts. After the samples were processed by homogenization and decontamination, acid-fast staining and culture into Löwenstein-Jensen medium were performed. Acid-fast bacilli isolated from culture medium were identified by using conventional methods, polymerase chain reaction (PCR)-RFLP (Restriction Fragment Length Polymorphism) and INNO-LIPA Mycobacteria methods. NTM were identified from 4.9% (5/101 ) of water samples and 0.8% (1/124) of soil samples by culture and PCR. No NTM were detected In the raw milk samples. Three of the NTM strains isolated from water samples were defined as Mycobacterium chelonae type III and two as Mycobacterium kansasil type II. One NTM strain isolated from soil was defined as Mycobacterium fortuitum. It was of note that two of the five NTM positive water samples were tap water samples collected from hospitals. It was concluded that NTM colonization/contamination of water and environment in the hospitals was a potential risk factor in terms of nosocomial infections. Thus surveillance cultures of the water systems and the medical devices in the hospital are necessary to fix the source of NTM, to identify and type the strains and to establish effective control measures such as sterilization, disinfection, maintenance and modernization of water systems.