To determine the utility of Southern blot analysis for fine-needle aspiration samples, the authors prospectively analyzed immunoglobulin, T-cell receptor, c-myc, and bcl-2 gene rearrangements in 27 cases of known or suspected lymphoma. Adequate DNA for analysis was obtained from 20 of 27 cases (74%), including 18 of 22 (82%) with non-Hodgkin's lymphoma (NHL). Patients whose tumors showed sclerosis, cellular degeneration, or necrosis yielded inadequate DNA. Of the 18 NHLs with successful Southern blot studies, 17 tumors had a B-cell lineage and one was an adult T-cell leukemia/lymphoma; clonal integration of the human T-cell leukemia virus I (HTLV-I) genome was present in the latter case. Four cases had bcl-2 rearrangements and two had c-myc rearrangements. One patient with follicular small cleaved cell NHL that evolved to a small noncleaved cell NHL had coexisting bcl-2 and c-myc rearrangement in the aspiration specimen of the high-grade NHL, suggesting sequential bcl-2 and c-myc activation during the tumor's progression. Southern blot analysis is a useful technique for establishing tumor cell lineage, clonality, and the presence of oncogene rearrangements in fine-needle aspiration specimens of NHL.