OBJECTIVE To assess the feasibility and role of fine needle aspiration biopsy (FNAB) as a diagnostic tool in children with neck masses. METHODS Retrospective chart review. METHODS Tertiary care children's hospital. METHODS Consecutive series of 71 children with a head and neck mass who underwent FNAB as the primary diagnostic modality. METHODS FNAB was performed and interpreted by a pediatric cytopathologist. Rapid on-site analysis was performed to allow immediate assessment of specimen adequacy and to attain a preliminary diagnosis, after which routine cytologic staining was performed. Flow cytometry was performed on cytological specimens when malignancy was suspected, and open biopsy was performed when the cytologic diagnosis was in question. METHODS Technical feasibility of FNAB in children, complications, cytopathological diagnoses, accuracy of rapid on-site analysis, need for subsequent diagnostic evaluations, clinical outcomes and follow-up. RESULTS Mean age was 8.4 years (S.D. 5.3 years), with mean follow-up of 4.1 months (S.D. 9.6 months). FNAB was performed under general anesthesia in 54 cases (76%). There were no technical complications. On-site rapid interpretation was completed in 55 cases, 18/55 confirmed adequacy of specimen only, 37/55 yielded a preliminary diagnosis, and in 34/37 cases, was same as final cytopathologic result. Overall, FNAB biopsy demonstrated 64 benign lesions, 3 malignant diagnoses, 2 follicular thyroid neoplasms, and 2 non-diagnostic specimens. FNAB was the only pathological test performed in 54 (76%) cases. The most common diagnosis was reactive lymphoid hyperplasia (n = 39), followed by benign granulomatous disease (n = 8). Flow cytometry was performed on 7 specimens (non-diagnostic in 5, negative for malignancy in 2). Of the 15 cases with surgical specimens, 3 revealed a pathologic diagnosis different from initial FNAB. There were no cases in which FNAB missed a malignancy, and there were 2 cases where FNAB suggested malignancy, with benign disease subsequently found on open biopsy. CONCLUSIONS Fine needle aspiration biopsy is developing into a feasible option in diagnosing pediatric neck masses, with its main advantage being its minimally-invasive nature and avoidance of an open surgical procedure for benign persistent lymphadenitis. On-site rapid interpretation can be used successfully to confirm specimen adequacy and to give an accurate preliminary diagnosis for concerned parents. Issues to consider include the need for a specialized pediatric cytopathologist familiar with pediatric differential diagnoses, the need for general anesthesia in many cases, and the possibility of inaccurate diagnosis requiring an open procedure.