Human monocytes express on their plasma membrane relatively large number of CD14 molecules, known to play a crucial role in the lipopolisaccharide (LPS)-mediated cellular activation. Indirect data (J. Biol. Chem. 270 (1995) 9904) suggest that not all of these CD14 molecules participate in LPS-signaling, but the importance of these spare receptors and the exact number of CD14 involved in activation upon different LPS-stimuli is not known. Using different concentrations of a blocking anti-CD14 monoclonal antibody (mAb 60bca) we created monocytes with graded amounts of CD14. The exact number of occupied and free receptors was quantitated by flow cytometry and special mAb-labeled standard beads. The number of free CD14 molecules per monocyte in the presence of 10, 3.33, 0.73, 0.25 and 0.041 microg/ml mAb was 0, 13,100, 49,300, 97,700 and 165,900. Stimulation of these partially blocked monocytes with 0.1, 1, 10 and 100 ng/ml ReLPS in the presence of 3% human serum revealed that already 13,100 and 97,700 CD14 molecules provided a maximal Tumor necrosis factor alpha (TNFalpha) mRNA response using 100 and 10 ng/ml ReLPS, while the activation totally depended on the number of available CD14 molecules in the case of 1 and 0.1 ng/ml ReLPS. Our data imply that the number of CD14 molecules available for LPS-binding influence the cellular response. In the presence of higher concentrations of LPS only fractions of CD14 participate in the cell activation, while the presence of the spare receptors enhance the sensitivity against lower LPS amounts.