We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.