CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2) have high sequence similarity and overlapping chemokine ligand profiles. Residue positions 3.32 and 7.39 are critical for signal transduction in the related CXCR4, and in these positions CXCR1 and CXCR2 contain oppositely charged residues (Lys^3.32 and Glu^7.39). Experimental and computed receptor structures reveal the possible formation of a salt bridge between transmembrane (TM) helices 3 and 7 via these two residues. To investigate the functional importance of Lys117^3.32 and Glu291^7.39 in CXCR1, along with the flanking Glu118^3.33, we performed a signaling study on 16 CXCR1 mutants using two different CXCL8 isoforms. While single Ala-mutation (K117^3.32A, E291^7.39A) and charge reversal (K117^3.32E, E291^7.39K) resulted in nonfunctional receptors, double (K117^3.32E-E291^7.39K) and triple (K117^3.32E-E118^3.33A-E291^7.39K) mutants rescued CXCR1 function. In contrast, the corresponding mutations did not affect the CXCR2 function to the same extent. Our findings show that the Lys^3.32-Glu^7.39 salt bridge between TM3 and −7 is functionally important for CXCR1 but not for CXCR2, meaning that signal transduction for these highly homologous receptors is not conserved.