We aimed to provide evidence that blood monocytes belonging to all subsets predominantly circulate in constant and usually reversible interactions with platelets, which are predominantly [Ca2+] dependent. The proportions of monocyte-platelet aggregates (MPAs) attributable to individual monocyte subsets in fresh and promptly processed heparin-anticoagulated blood from 10 healthy subjects (median age 35 years, 50% male) were analysed by flow cytometry and compared to samples anticoagulated with a potent [Ca2+] chelator, ethylenediaminetetraacetic acid (EDTA). Additional experiments with [Ca2+] depletion or supplementation were also performed. Monocytes subsets were defined as CD14++CD16-CCR2+ cells (Mon1), CD14++CD16+CCR2+ cells (Mon2) and CD14+CD16++CCR2- cells (Mon3). Vast majority of monocytes showed aggregation with platelets in heparinised samples, but most monocytes were free of platelets when EDTA was used (p<0.001 for all subsets). Addition of the heparinised blood to EDTA-containing vacutainers reduced the proportion of MPAs to values seen in the directly EDTA-anticoagulated blood (p=0.005 for all subsets). Supplementation with CaCl2 resulted in dose-dependent increase in MPAs (p<0.001 for all subsets). Although the overall trend for the monocyte-platelet interactions was applicable to all monocyte subsets, the proportion of MPAs in heparinised samples was lowest for Mon3 (p<0.0001). In contrast, Mon3 showed the highest proportion of MPAs in EDTA-anticoagulated samples (p=0.004). In healthy subjects monocytes circulate in constant, but predominantly reversible and [Ca2+]-dependent aggregation with platelets. These observations may reflect a complex involvement of platelets in regulation of monocyte activity.