BACKGROUND: The 2007 World Health Organization (WHO) guideline to diagnose smear-negative tuberculosis (TB) in HIV-prevalent settings was mainly based on expert advice and therefore requires evaluation in real life situations. METHODS: In 2009, this guideline was introduced at the ALERT hospital in Ethiopia. From October 2009 to January 2011, the accuracy of the guideline was evaluated using Mycobacterium tuberculosis culture positivity as reference standard in HIV positive TB suspects. RESULTS: A total of 459 TB suspects were enrolled during the study period; 336 (73.2%) were HIV positive. Acid fast bacilli sputum smear microscopy was done for 74.7% (251/336) HIV positive TB suspects; 94.4% (237/251) were smear negative. A chest X-ray was performed in 92.8% (220/237) and a Mycobacterium tuberculosis culture in 63.7% (151/237). The median TB diagnostic delay for smear negative cases was 3 days (interquartile range 3-4 days). Of the 75 patients diagnosed with smear negative pulmonary TB, 89. 4% (67/75) were diagnosed by chest X-ray, 9.4% (7/75) by culture and 1.3% (1/75) by clinical suspicion only. In 147 smear negative TB suspects Mycobacterium tuberculosis culture and chest X-ray results were available. Among these 147 patients, the sensitivity of the chest X-ray to diagnose smear negative TB in HIV-positive TB suspects was 53.3% (95% CI: 26.7-78.7); the specificity 67.4% (95% CI: 58.7-75.3). CONCLUSION: The 2007 WHO diagnostic algorithm for the diagnosis of smear negative TB is likely to reduce the diagnostic delay and therefore decrease morbidity and mortality of TB in a HIV prevalent settings like Ethiopia.