Purpose: Mediastinal lymphadenopathy (ML) is a cause for concern, especially in patients with previous malignancy. EUS-guided FNA has significantly expanded the diagnostic capability of EUS in GI disorders including tissue evaluation of ML. Therefore, we sought to investigate the use of EUS-guided- FNA with immunostains in patients being evaluated for ML. Methods: We prospectively analyzed adult patients with ML of unknown origin who underwent EUS guided-FNA at a tertiary referral center between September 2000 and May 2007. EUS-guided FNA was performed with linear echoendoscopes and 22-gauge needles. On-site and final evaluation was performed by experienced cytologist, in addition to referral to special immunostains if necessary. Patients' demographics were documented, as well as lymph node (LN) characteristics. Results: One-hundred and nineteen patients were included, and a total of 138 mediastinal LN were sampled. Mean age of patients was 54.4 years (SD 16.6), 53.8% were men (64 patients) and 77.3% were white (92 patients). Of the 119 subjects, prior malignancy was present in 44.5% (61 patients). Prior chest CT was performed in 87% (100 patients); PET scan in 38% (44 patients, median SUV 6, IQR 3-10, range 2-52 n=22). Prior LN biopsy was performed in 24 patients (20.3%). Site of examined LN: 101 subcarinal (76.5%), 11 lower paraesophageal (8%), 8 aorto-pulmonary window (6%). The median ML diameter was 28 mm × 13mm. Sharp borders were seen in 28 (23.7%), round shape in 21 (15.9%), hypoechoic appearance in 53 (40.1%). Mean number of passes was 3.83, SD1.34. FNA was read on-site as malignant 21 (16.1%), benign 100 (76.9%), suspicious 6 (4.6%), atypical 3 (2.3%). Final FNA read was malignant in 28 (21.8%), benign 98 (76%), suspicious 3 (2.3%). Sixty-four LN were deferred for additional studies; 22 for immunostains, 26 for GMS stain and 22 for flow cytometry. The sensibility, specificity and overall accuracy of on-site read cytology was, respectively, 95%, 94% and 86%. Metastatic malignancies disclosed included Hodgkins lymphoma (2), NHL (5), lung (4), melanoma (1), breast (6), colon (3), renal (2), endometrial (1), Fallopian tube (1), hepatoma (1), unknown primary site(2).The significant predictors of malignancy in FNA were larger short axis(OR 1.08, 95%CI 0.99-1.18, P = 0.093), prior malignancy, (OR 12.90, 95%CI 2.90-57.62, P = 0.001) and presence of sharp borders (OR 5.44, 95%CI 1.43-20.70, P=0.013). Conclusion: EUS-guided FNA with additional ancillary studies and immunostains is useful in disclosing metastatic lymphadenopathy to the mediastinum from a variety of neoplasms. Predictors of malignancy include prior history of malignancy, larger short axis and presence of ML sharp borders.