<b><i>Background:</i></b> Few prospective studies have evaluated the role of endobronchial needle aspiration in diagnosing central airways neoplasms. Rapid on-site evaluation has long been used in transbronchial needle aspiration of adenopathies and peripheral lesions, but its role in sampling central malignancies has not been substantiated yet. <b><i>Objectives:</i></b> In this study we evaluated if endobronchial needle aspiration may increase the sensitivity of bronchoscopy for diagnosing central airways neoplasms when added to conventional diagnostic methods (forceps biopsy, brushing and bronchial washing), and if rapid on-site evaluation may be beneficial in patients undergoing endobronchial needle aspiration. <b><i>Methods:</i></b> 125 patients (77% males, aged 70 ± 7 years; mean ± SD) with central lung cancers were randomized to undergo bronchoscopy including conventional diagnostic methods and needle aspiration, with or without rapid on-site evaluation, stratifying the patients on the basis of the neoplasm growth pattern (exophytic and submucosal/peribronchial disease). <b><i>Results:</i></b> Needle aspiration significantly increased the sensitivity of bronchoscopy when added to conventional methods (from 76 to 91%; p < 0.001), primarily resulting from differences in submucosal/peribronchial diseases (68 vs. 90%; p < 0.001) and independently from the presence of rapid on-site evaluation; needle aspiration guided by rapid on-site evaluation guaranteed a higher improvement in bronchoscopy sensitivity than conventional needle aspiration (98 vs. 84%, respectively; p = 0.004). Needle aspiration guided by rapid on-site evaluation showed a significantly higher sensitivity than the conventional method (97 vs. 76%, respectively; p = 0.001). <b><i>Conclusions:</i></b> Needle aspiration increases the sensitivity of bronchoscopy in diagnosing central airways malignancies when added to conventional diagnostic methods, with a further significant improvement when guided by rapid on-site evaluation.