The genetic events involved in the transformation of chronic lymphocytic leukemia (CLL) to Richter's syndrome (RS) are poorly understood. Frequently large cells are seen in the bone marrows of patients with CLL and evidence of RS. Using a laser-capture microdissection we analyzed small and large leukemic bone marrow cells from 19 patients with RS for loss of heterozygosity (LOH) on chromosome 11 (D11S2179 at the ATM gene), 17 (D17S938 and D17S1852 at the TP53 site), and 20 (Plc1, D20S96, D20S110, and D20S119). Megakaryocytes were also isolated and used as a control for normal cells. Four of 15 (27.7%) informative cases showed LOH in small cells in the ATM gene while seven (46.7%) showed LOH in large cells. Six of 15 (40%) informative cases had LOH in chromosome 17 in small cells, and eight (53%) showed LOH in large cells. Eleven of 19 informative cases (61.1%) showed LOH in chromosome 20 in large cells, and eight (42.1%) showed LOH in small cells. RS cases with LOH at chromosome 20 were associated with marginally shorter survival rates (P = 0.08). Our data suggest that there are significant molecular differences between large and small cells in patients with CLL. Further analysis of the genes on these chromosomes may provide new insight into our understanding of the transformation of small CLL cells to large (Richter) cells.