Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.