An orthogonal tryptophanyl–transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA ^Trp ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{mutRNA}}_{{\mathrm{UCA}}}^{{\mathrm{Trp}}}\end{equation*}\end{document} ) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA ^Trp was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{mutRNA}}_{{\mathrm{UCA}}}^{{\mathrm{Trp}}}\end{equation*}\end{document} gene was inserted between the 5′ and 3′ flanking sequences of the tRNA ^Trp-1 gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{mutRNA}}_{{\mathrm{UCA}}}^{{\mathrm{Trp}}}\end{equation*}\end{document} and no endogenous mammalian tRNAs. Similarly, the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{mutRNA}}_{{\mathrm{UCA}}}^{{\mathrm{Trp}}}\end{equation*}\end{document} is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy- l -tryptophan. The resulting mutant \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{BsTrpRS}}{\mathrm{-}}{\mathrm{mutRNA}}_{{\mathrm{UCA}}}^{{\mathrm{Trp}}}\end{equation*}\end{document} pair allows the efficient and selective incorporation of 5-hydroxy- l -tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.