We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14(++)CD16(-) and the CD14(+)CD16(++) monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14(+)CD16(++) monocyte gate. For these CD14(+)CD16(++) monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14(+)CD16(++) monocytes (45.4 +/- 13.5 cells/microl) compared with males (59.1 +/- 20.3 cells/microl) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14(+)CD16(++) monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease.