Thioamide substitutions in the backbones of proteins can modulate their structure and thermostability, or serve as spectroscopic probes in fluorescence quenching experiments. Using native chemical ligation, we have produced the first examples of a protein (calmodulin) containing two thioamides. Dithioamide variants were made to explore the effects of combining stabilizing, neutral, and destabilizing single thioamide substitutions. One of the dithioamide calmodulin variants exhibited stabilization greater than any monothioamide variant, although the effect could not easily be anticipated from the results of single substitutions. Each of the calmodulin variants retained the ability to bind a target peptide, and the dithioamide proteins exhibited an increase in fluorescence quenching of tryptophan relative to their single thioamide counterparts. These results show that multiply thioamidated proteins can be synthesized, and that properly placed thioamides can be used to increase protein thermostability or enhance fluorecsence quenching in peptide binding experiments.