The thromboplastin synthesis of the human monocytoid cell line U-937 and its two subclones designated U-937-3 and U-937-4 has been studied. U-937-4 seems by several functional criteria to represent a more advanced stage of monocyte differentiation than the original U-937. U-937-3 appears to be arrested at an even more immature stage than the original population. The basal thromboplastin activity was higher in U-937-4 than in U-937-3 or U-937 cells (7.0 +/- 1.9 (SEM), 1.0 +/- 0.2 and 1.6 +/- 0.6 units/mg protein, respectively) although not as high as in human normal monocytes (14.1 +/- 2.4). The thromboplastic expression of the two clones was maximal when cells were in logarithmic growth. Both clones responded with a weak to moderate thromboplastin synthesis upon addition of stimulants like phytohaemagglutinin (PHA), immune complexes or endotoxin. Thromboplastin production was also potentiated in the presence of lymphocytes. The supporting effect of lymphocytes was strong in the case of U-937-3 as well as in U-937 cells, but less pronounced in U-937-4 cells as it also is in human monocytes. The thromboplastin response after PHA stimulation was more rapid in U-937-4 cells (maximal after 4-8 h) than in U-937 or U-937-3 cells (12-16 h). Human monocytes also responds quickly to PHA (maximally 4 h). Total phospholipid content and the relative distribution of individual phospholipids were essentially similar in U-937-3, U-937-4 and U-937. With regard to thromboplastin production, U-937-4 cells seem to be more monocyte-like than the more immature cells U-937-3 and U-937. It is concluded that thromboplastin seems to be a useful marker for monocyte differentiation.