Crohn's disease (CD) is a gastro-intestinal inflammatory disease with uncertain origin. Autoimmune response and/or infectious etiologies have been proposed as possible causes. A role for Mycobacterium avium subsp. paratuberculosis (Map) in CD has been hypothesized. However, the role of Map in disease induction is still in debate. The aim of the present study is to obtain indications on Map isolation from CD patients living in an area of Northern Italy characterized by the presence of a high concentration of paratuberculosis infected cattle herds. In the area of concern, a mean annual incidence rate of 3.4 CD patients per 100.000 inhabitants has been reported by a study which considered a 4 years observation period. Since now, the present study considered 112 human intestinal mucosal biopsy specimens, deriving from 8 patients with CD, 13 patients with ulcerative colitis (UC), 9 patients with undefined inflammatory bowel disease (IBD) and 80 healthy patients. For each patient, six bioptic tissue pieces from ileo-colon region were collected and processed for Map isolation. Culture was performed with BacT/ALERT 3D system (bioMérieux) using a Middlebrook 7H9-based liquid medium. The suitability of the system to grow Map was previously assessed by cultivation of the ATCC 19698 strain. None of the biopsies were Map culture-positive. An alcohol-acid fast organism was recovered from a healthy control patient. The isolate was negative when tested by IS900-PCR and able to grow in absence of mycobactin J. In conclusion, the preliminary results obtained do not indicate the presence of Map in subjects examined. However, a higher number of subjects and an extension of incubation times is needed for a better evaluation of the presence of Map in Crohn's disease patients by this culture method.