The stimulation of steroidogenesis by adrenocorticotropic hormone (ACTH) and adenosine 3'-5' monophosphate (cyclic-AMP) was investigated in in vitro systems. Temperature is one of the most important elements for cell culture and affects various biological cell functions. In this study, the effects of various temperatures (32-43°C) on the stimulation of steroidogenesis by ACTH and cyclic AMP in the murine adrenal tumor cell (Y-1) were investigated. After Y-1 cells were cultured at 32-43°C for 48 hours, most of the population survived. Y-1 was insensitive to high temperature during 48 hours. When cells were incubated at 32°C, steroids in the medium could not be detected, but at 33-39°C, steroid amounts increased linearly with increasing temperature. At 41°C or higher, steroid secretion decreased drastically. Although the stimulation of steroidogenesis by ACTH was accelerated sharply at 33°C, stimulation by cyclic AMP may be gradually increased until 39°C. These data suggest that this may be caused by different action sites of ACTH and cyclic AMP. More studies indicated that ACTH binds to the plasma membrane and activated adenyl cyclase, but cyclic AMP function required binding to the specific cytosol receptor.