Abstract The human HSP90 gene family contains introns. There are two typical heat shock elements (HSE) in the first intron of human hsp90β gene. As detected by chloramphenicol acetyl transferase (CAT) reporter activity assays, the HSE-containing intron is essential in maintaining high constitutive expression and is critical for heat shock inducibility of the human hsp90β gene. Cellular heat shock factor 1 (HSF 1) shows much higher binding affinity toward the intronic HSEs in comparison to an atypical HSE in the 5′ flanking sequence. Novel initiation sites found in the first intron probably also contribute to constitutive and heat-inducible expression of the hsp90β gene in Jurkat cells.