NhaA, a Na(+)/H(+) antiporter critical for pH and Na(+) homeostasis in Escherichia coli, as well as other enterobacteria and possibly Homo sapiens, was modified for fluorescence spectroscopy by constructing a functional Trp-less NhaA mutant. Purified Trp-less NhaA lacks the Trp fluorescence emission characteristic of the wild type, thereby providing a background for studying structure-function relationships in NhaA by site-directed Trp fluorescence. Two single-Trp variants in the Trp-less background (F136W and F339W) were constructed. The mutants grow on selective media, have antiport activities that are similar to Trp-less NhaA, and exhibit Trp fluorescence with three different reversible responses to Li(+), Na(+), and/or pH. With single Trp/F136W, a pH shift from pH 6.0 to 8.5 induces a red shift and dramatically increases fluorescence in a reversible fashion; no effect is observed when either Na(+) or Li(+) is added. In marked contrast, with single Trp/F339W, changes in pH do not alter fluorescence, but addition of either Na(+) or Li(+) drastically quenches fluorescence at alkaline pH. Therefore, a Trp at position 136 specifically monitors a pH-induced conformational change that activates NhaA, whereas a Trp at position 339 senses a ligand-induced conformational change that does not occur until NhaA is activated at alkaline pH.