When cells are exposed to elevated temperatures, transcription of a small set of genes, the heat-shock genes, is activated. This response is mediated by a short DNA sequence, the heat-shock element (HSE), which is thought to be the binding site for a specific transcription factor. Studies with Drosophila show that this protein binds to HSEs only in heat-shocked cells, suggesting that changes in factor binding are responsible for gene activation. We have investigated the properties of HSE-binding proteins from yeast and HeLa cells. In HeLa cells, binding activity is present only after heat shock. In contrast, control and heat-shocked yeast cells yield the same amount of HSE-binding activity; however, the mobility of protein-HSE complexes on polyacrylamide gels is altered following heat shock. This mobility difference can be significantly reduced by treatment of crude extracts with phosphatase. We propose that the yeast heat-shock factor binds constitutively to DNA but only activates transcription after heat-induced phosphorylation.