The immune system relies on chemokines to orchestrate cell migration during immune surveillance and inflammation. In all of these contexts, chemokines interact with seven-transmembrane chemokine-type G protein-coupled chemokine receptors. Chemokine receptors have emerged as promising drug targets in a wide range of inflammatory, autoimmune and infectious diseases. In my thesis I studied the key determinants for chemokine receptor engagement and activation using two model receptor systems - CCR5/CCL5 and CCR9/CCL25 – with the ultimate goal of better targeting these axes in disease. Chemokine ligands engage the receptor via a “two-site” mechanism. The chemokine core region binds to the receptor N-terminus and extracellular loops (CRS1) regulating binding affinity and specificity; the flexible chemokine N-terminus binds to the transmembrane domain of the receptor (CRS2), promoting receptor activation. The CRS1 interaction is dominated by an electrostatic interaction between a conserved positively charged groove on the chemokine core region and negative charges on the receptor N-terminus, largely provided by post-translational modifications (PTMs) such as tyrosine sulfation and O-glycan sialylation. In my thesis I leveraged CCR5 existing molecular toolbox including monoclonal antibodies and highly potent chemokine analogs developed in our laboratory, to investigate the impact of N-terminal PTMs on CCR5 cell surface heterogeneity and ligand-receptor interactions at the level of CRS1. Next, I investigated CRS2 interactions and activation determinants in the CCR9/CCL25 chemokine receptor.