Human monocytes are subdivided into classical, intermediate and non-classical subsets but there is no unequivocal strategy to dissect the latter two cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14+CD16++ non-classical monocytes and slan-negative CD14++CD16+ intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of MHC class II genes, while a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering the slan-positive non-classical monocytes cluster with monocytes and are clearly distinct from CD1c-positive dendritic cells. In clinical studies we show a selective increase of the slan-positive intermediate monocytes to >100 cells/μl in patients with sarcoidosis and a 5-fold depletion of the slan-positive monocytes in patients with hereditary diffuse leukodystrophy with axonal spheroids (HDLS), which is caused by macrophage colony stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.