A bioradioimmunoassay is described for determining adrenocorticotropic hormone (ACTH) levels in biological fluids. Slices of guinea pig's adrenal gland surviving in vitro and depleted of their cortisol content were used. When challenged with ACTH, the slices release newly synthesised cortisol into the medium which is then determined by radioimmunoasssay. A log-linear relationship was evident between added ACTH and the cortisol measured in the medium. Unlike previous methods, the sensitivity of this assay allows the determination of 0.5 femtogram (10/sup -15/g) of ACTH. A good correlation was observed when rat plasma ACTH levels were determined by the present bioassay method and by an ACTH radioimmunoassay. Mean values of plasma ACTH determined by the present method are shown for man, child, guinea pig, rabbit, rat, and hamster.