Geldanamycin (GA), a specific inhibitor of the chaperoning function of heat-shock protein 90 (Hsp90), has been shown to mimic heat shock (HS) in inducing expression of Hsp90, Hsp72 and other Hsps in unstressed mammalian cells. In the present study, intra-cerebral treatment of goldfish with GA (at a dose of 0.1 μg/g-body weight) diminished basal Hsp90 level to a 30–40% level in the brain, without affecting the basal Hsp72 level, as assayed 28–48 h after treatment. Whole-body exposure to HS significantly increased Hsp90 level in GA-untreated fish but not in GA-treated fish, while it significantly increased Hsp72 level in both GA-untreated and -treated fish. In both GA-untreated and -treated fish, plasma cortisol (PC) levels increased considerably 4 h after HS and then decreased in a time-dependent manner to the control levels 24 h after HS, showing no evidence of a GA effect on the time course of PC level. These results suggest that in the brain of goldfish, Hsp90 may not be involved as a key factor either in regulating Hsp72 expression both before and after HS or in the feedback regulation of HS-increased PC level, and support the idea that GA can be used in fish brain as a tool in elucidating the role of Hsp90 in complicated, Hsp-mediated biological processes.