CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (-GalCer, C20 and OCH12) for 4 and 16hr. Whereas CD1d--GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to similar to 10000nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to similar to 30000nm(2). Moreover, the smallest size of recognition spots was about 25nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions. Copyright (c) 2013 John Wiley & Sons, Ltd.