A simple one-step procedure for the determination of thymosin beta 4 in whole blood, various blood corpuscles, plasma and serum by reversed-phase high-performance liquid chromatography (HPLC) is described. The blood cells were isolated by a Ficoll density gradient technique. The isolated cells were lysed and deproteinized with perchloric acid in the presence of an internal standard. The supernatant solution was neutralized by potassium hydroxide solution and thymosin beta 4 was measured by HPLC without further manipulations. The following amounts (fg per cell) were determined: polymorphonuclear leukocytes (409), mononuclear leukocytes (267) and platelets (22.3). Erythrocytes contained no thymosin beta 4. The average concentration of thymosin beta 4 in an extract of whole blood was 16.3 micrograms/ml. The concentration in plasma was less than 1% of that value. However, if serum was prepared and not immediately removed from the clotted blood, the concentration of the peptide increased from 0.04 to 2.1 micrograms/ml within 24 h. The biological significance of the findings is discussed.