Abstract8‐Prenylnaringenin and isoxanthohumol are prenylflavonoids found in the hop plant, Humulus lupulus (Cannabaceae), which is traditionally used to add bitterness and flavor to beer. Flavonoids have previously been reported to exert endocrine disrupting actions. Therefore, we investigated the effects of 8‐prenylnaringenin and isoxanthohumol on steroidogenesis activated by human chorionic gonadotropin (hCG) in primary cultures of rat Leydig cells at different stages of their development. The present study is the first to demonstrate that the prenylflavonoids 8‐prenylnaringenin and isoxanthohumol exert complex maturation‐dependent effects on Leydig cell steroidogenesis. Those compounds inhibited hCG‐stimulated androgen production by Leydig cells at all stages of their development, a process that was associated with the reduced ability of the cells to produce cAMP. However, these same compounds up‐regulated hCG‐activated StAR expression in progenitor (PLC) and immature (ILC) but not adult types of Leydig cells (ALC). Further, 8‐prenylnaringenin and isoxanthohumol were not able to suppress androgen production activated by an exogenous analog of cAMP, (Bu)2cAMP, in ALC and ILC but synergistically stimulated steroidogenesis in PLC. Our data suggest that 8‐prenylnaringenin and isoxanthohumol affect cAMP‐dependent cellular processes up‐stream transport of cholesterol into mitochondria. Copyright © 2010 John Wiley & Sons, Ltd.