Neutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca2+ mobilization by the CXCR2-specific chemokine, growth-related oncogene α (Groα), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene α did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca2+ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross- desensitization of its Ca2+ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca2+ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C β3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation.