Sulfotyrosines contribute to a number of critical extracellular protein-protein interactions, including the association of the HIV-1 envelope glycoprotein with the HIV-1 coreceptor CCR5, a similar association between the Duffy binding protein of Plasmodium vivax and the Duffy antigen/receptor for chemokines, between complement components C5a and C3a and their respective receptors, and between many CC- and CXC-chemokines and their receptors. In addition, the antigen-combining regions of a number of human antibodies include sulfotyrosines that are necessary for antigen recognition. The study of sulfotyrosines requires an array of techniques, each with its advantages and limitations. These include modulation of tyrosyl-protein sulfotransferase activity in mammalian cell lines, production of tyrosine-sulfated peptides with direct chemical synthesis or enzymatic addition of sulfate to tyrosines in cell-culture or cell-free systems, and use of a novel tRNA/tRNA-synthetase pair capable of introducing sulfotyrosines at specific sites into bacterially expressed proteins. Here we describe the use of these various approaches to study the role of tyrosine sulfation of chemokine receptors in ligand binding and HIV-1 entry.