Background: Vaginal fine-needle aspiration (FNA) is infrequently performed to assess palpable lesions. We perform the first multi-institutional study to evaluate this procedure. Methods: We retrospectively reviewed vaginal FNAs performed at two institutions for the past 27 years. Clinical, cytological and histological data were reviewed and tabulated. Results: We identified 43 specimens from 39 patients (mean age 56 years, range 18-86 years). Twenty four patients (62%) had prior malignancies from the following sites: gynecologic tract (22), bladder (1), and breast (1). Twenty four specimens were malignant, 18 were benign (including eight cases from patients with prior malignancy) and one was unsatisfactory. Of 28 FNA specimens from patients with a malignant history, 18 (64%) were positive for malignancy. The most common malignancies were metastatic ovarian carcinoma (50%), squamous cell carcinoma (25%), and uterine cancer (17%). Mean time to metastasis/recurrence was 16 months and was longest in patients with ovarian metastasis (26 months) compared to other malignancies (P = 0.002). The most common benign diagnoses were cysts (33%) and inflammation (22%). In 27 cases with histological correlation, there were 20 true positives, six true negatives and one false negative (sensitivity =95%, specificity =100%). Seven patients had a recent Pap test with two true positives, two true negatives, and three false negatives (sensitivity = 40%, specificity = 100%). Conclusion: Vaginal FNA is usually performed to rule out a secondary malignancy, often of ovarian origin. Vaginal metastases from extra-gynecologic sites are rare. FNA is both highly sensitive and specific and may be a safe and effective alternative to biopsy. Diagn.