Thrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well‐known chemotactic agents such as fMet‐Leu‐Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL‐60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP‐α‐thrombin, as well as the thrombin‐derived chemotactic peptide CB67–129, elicits a dose‐dependent chemotactic response in HL‐60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL‐60/mono), whereas no such response is evident in either undifferentiated HL‐60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL‐60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte‐differentiated cells. In HL‐60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15–20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL‐60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL‐60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL‐60/mono (but HL‐60/gran nor undifferentiated HL‐60) are capable of significant specific 125‐I‐labeled α‐thrombin‐binding (ka∼20 nM), and possess an estimated 400,000 thrombin‐binding sites per cell. Our finding further suggest that the thrombin response of HL‐60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.