To analyze the value and limitations of flow cytometry (FCM) in the investigation of patients with lymphadenopathy, a retrospective study of 196 patients, referred for fine-needle aspiration (FNA) cytology, was carried out in Canberra, Australian Capital Territory, Australia, between 1992-1997. Complete cytological, flow-cytometric, and outcome (clinical and histological) data were available on all the cases. The FNA appearances were read in conjunction with FCM findings. The following cytological categories were recognized: benign, 78 cases (39.8%); indeterminate, 9 cases (4.6%); and malignant, 109 cases (55.6%). None of the 78 cytologically benign cases had malignant outcome. All 109 cytologically malignant cases had malignant histology, and 8/9 of the cytologically indeterminate FNAs had malignant histology. The cytologically malignant category contained 106 B-cell lymphomas and three T-cell lymphomas. All 65 B-cell lymphomas with K light chain predominance had K/L ratio greater than 3/1, and all 34 B-cell lymphomas with L light chain predominance had an L/K ratio greater than 2/1. Clonality was therefore established for K/L and L/K at 3/1 and 2/1, respectively. When K/L and L/K ratios were below these figures (7 cases), other parameters, including the proportion of CD20 and the dual expression of CD19/CD10 and CD20/CD5, were used to determine the nature of the aspirate. In the B-cell lymphomas without demonstrable light chain restriction, CD20 positivity in excess of 85%, CD19/CD10 positivity of more than 18%, or CD20/CD5 positivity greater than 35% were independently diagnostic of B-cell lymphoma. In the T-cell lymphomas, greater than 90% of the cells were T cells, and aberrant T-cell antigen expression with loss of at least one pan-T-cell antigen was detected. In conclusion, the sensitivity of diagnosis of malignancy, false-negative rate, and predictive value of malignant diagnosis with combined FNA cytology and FCM were 99%, 0%, and 100%, respectively.