Downregulation of HLA-DR on monocytes has been proposed as an adverse predictive factor in sepsis. In those studies, determinations have been performed by dual color flow cytometry, using anti-CD14 and anti-DR monoclonal antibodies (MoAbs). In our hands, this method proved unreliable. Therefore, we scheduled this study to provide the most appropriate method for the flow cytometric quantitation of DR-monocytes, in order to examine their possible diagnostic significance in neonatal sepsis. To this aim, repeatitive measurements were performed in blood samples obtained from normal individuals, premature neonates and infectious conditions followed by monocytosis (20-25 from each category), using the following flow cytometric approaches: [a] Dual color labelling of CD14/HLA-DR using alternatively fluorescein (FITC) and phycoerythrin (PE) probes, in order to examine the possible effect of steric hindrance and/or quenching phenomena. [b] One color HLA-DR labelling in the monocyte scatter gate to further exclude the possibility of such interventions. [c] One color HLA-DR labelling in a gate for monocytes, obtained using the combination of CD3-CD19-PE/CD15-FITC MoAbs. In this way, cells with variable DR expression, such as B, T lymphocytes and granulocytes, are excluded. A whole blood staining method with red cell lysis was used in all cases. It was observed that: (a) Both CD14-FITC/DR-PE and CD14-PE/DR-FITC combinations gave DR-monocyte values with very low repeatability and very high variability. The comparison of the values obtained by these combinations proved statistically very significant (paired-t-test, p<0.01). (b) One color DR labelling gave consistently higher DR-monocyte values compared to the dual labelling. (c) A dim CD14 monocyte subpopulation was systematically excluded from the DR study of monocytes as determined by the dual labelling CD14/DR method. This subpopulation also appeared with bright CD16 fluorescence. [d] By the CD3-CD19-PE/CD15-FITC gating method, the highest purity and recovery values of monocytes were achieved, while measurements were characterised by high repeatibility and very low variability. It is concluded that this final approach provides the most reliable estimation of DR-monocytes. Furthermore, only by this method it is possible to measure the fluorescence intensity and, therefore, to obtain an estimation of the number of HLA-DR molecules on monocytes, expressed in molecules of equivalent soluble fluorochrome (MESF) units. This can be achieved by adding a mixture of five populations of microbeads with different calibrated binding capacities(Quantum Simply Cellular, FCS).