Recently, we have shown that thrombin is a chemotaxin and growth-promoting agent for cells of the mononuclear phagocytic lineage. These activities are independent of thrombin's enzymatic activity. Unlike other chemotactic factors, thrombin is specific for monocytes and does not attract granulocytes. To further explore the cellular specificity we have used a human leukemia cell line HL-60 that is capable of in vitro differentiation toward either monocytes (HL-60/mono) following incubation with 1,25(OH)2D3, or granulocytes (HL-60/gran) following incubation with DMSO. In contrast to undifferentiated HL-60 cells or HL-60/gran, we find that HL-60/mono respond chemotactically to intact human alpha-thrombin, esterolytically inactive iPR2P-alpha-thrombin, and the thrombin-derived peptide CB67-129, previously shown to contain the thrombin chemotactic exosite. In addition, thrombin induces in HL-60/mono association of actin with the cytoskeleton and causes an increase in levels of free cytosolic Ca2+. These phenomena are well characterized as early events occurring concomitant with directed cell movement associated with exposure to chemotactic agents such as FMLP. Furthermore, in contrast to fibroblasts, both iPR2P-alpha-thrombin and the thrombin chemotactic peptide CB67-129 evoke dose-dependent [3H]TdR incorporation, protein synthesis, and cell replication in growth-arrested J-744 cells, a murine macrophage-like cell line. Limited tryptic digests of CB67-129 lose chemotactic activity but retain full mitogenic activity, demonstrating that as with PDGF, the sites on CB67-129 required for chemotaxis and mitogenesis are clearly dissociable. The mitogenic effects of the CB67-129 digest can be mimicked by a synthetic tetradecapeptide analogue of CB67-129 (residues 367-380) that includes the loop B insertion sequence, previously shown to be critical for thrombin's chemotactic effects. From these data, it is apparent that the loop B insertion is critical for thrombin's nonenzymic biological effects on cells, but additional sites are required for stimulation of cell movement.