Between 1980 and 2003, 13 patients (0.95% of all cases of tuberculosis) at a 600-bed university hospital in Madrid, Spain, were diagnosed with Mycobacterium bovis infection. All 13 cases occurred between 1994 and 1999; the mean age of the patients was 50 years (range 23-83 years), and 77% were males. Four (30%) patients were also positive for human immunodeficiency virus (HIV). The most frequent localisation of the disease was the lung (ten patients; 77%). Seven patients, including four HIV-positive patients who died, had multidrug-resistant M. bovis infection. No other patient died, including two HIV-negative patients with multidrug-resistant disease.