Interleukin-8 (IL-8 or CXCL8) interacts with two receptors CXCR1 and CXCR2 to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (~90% vs ~10%, respectively, after 5 min). The carboxyl-terminus of the receptors were shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of downregulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in RBL-2H3 cells and characterized for receptor binding, intracellular Ca2+ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization and, MAP kinase activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 for that of CXCR1 (B2ECLA) or aspartate-199 for its valine counterpart (BD199VA) delayed CXCR2 internalization similar to that of CXCR1. Replacing aspartate-199 for asparagine (BD199N), restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggests that aspartate-199 plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. BD199N internalized rapidly but migrated as a single phosphorylated form like CXCR1(~75 kDa) whereas B2ECLA and BD199VA showed a slow and a fast migrating form like CXCR2 (~45 and ~65 kDa) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptors internalization. Like CXCR1, BD199VA also induced sustained ERK activation and cross-desensitized Ca2+ mobilization to CCR5, relative to BD199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors are important in modulating their distinct rate of downregulation thereby signal length and post-internalization activities.