Temperature-sensitive (Ts) mutants are important tools for understanding the role of essential gene(s), but their molecular basis is not well understood. We use CcdB ( Controller of Cell Death protein B) as a model system to explore the effects of Ts mutations on protein stability, folding, and ligand binding. Previously isolated Ts CcdB mutants fall broadly into two categories, namely, buried site (<5% accessibility) and active site (involved in DNA gyrase binding). Several mutants from each category were characterized. It was found that buried-site Ts mutants had decreased stability and foldability, higher aggregation propensity, and, in most cases, reduced affinity for gyrase at both permissive and restrictive temperatures. In contrast, exposed, active-site Ts mutants of CcdB exhibited stability either higher than or similar to that of the wild type and weakened inhibition of DNA gyrase function and/or reduced affinity for gyrase at a higher temperature. At all temperatures, Ts mutations at exposed, active-site residues primarily decrease specific activity without affecting protein levels, while Ts mutations at most buried residues decrease both specific activity and protein levels. Ts phenotypes in both cases arise because total activity is decreased below the threshold required for survival at the restrictive temperature but remains above it at the permissive temperatures. For several mutants, Ts phenotypes were ameliorated upon overexpression of the trigger factor chaperone, suggesting that Ts phenotypes may result from mutational effects on in vivo protein folding rather than on protein stability. This study delineates the diverse factors that contribute to Ts phenotypes. These insights can facilitate rational design of Ts mutants.