Morphological and biochemical differences were demonstrated between prolymphocytic leukemia cells obtained from the spleen and peripheral blood of one patient. Peripheral blood prolymphocytes had consistently smaller nuclear-cytoplasmic ratios than did splenic prolymphocytes. Percoll gradient-purified prolymphocytes from the spleen synthesized abundant amounts of some membrane proteins which were hardly expressed by peripheral blood prolymphocytes. Peripheral blood prolymphocytes did not change their expression of membrane proteins during three days in culture. These findings are consistent with the view that prolymphocytic leukemia cells from the spleen exist, on the average, at an earlier stage of differentiation than do circulating leukemic cells, and that peripheral blood leukemic cells are frozen at a specific phase of differentiation.