The structural requirements in the ACTH molecule for evocation of the glycolytic response in suspensions of mouse adrenal cells were investigated by examining the effects of analogues containing modifications at positions 8, 9 and 10 and of peptides containing homologies with the amino-terminal segment of ACTH. Introduction of a nitrophenylsulphenyl (NPS) group into the tryptophan moiety at position 9 of ACTH(1-24) greatly reduced both the potency and the capacity for maximal glycolytic response. It also virtually abolished cyclic AMP formation. In contrast, the capacity for a maximal steroidogenic response remained unimpaired in the NPS derivative, although steroidogenic potency was reduced to 0.4% of that of ACTH(1-24). Replacement of the tryptophan moiety with phenylalanine had intermediate inhibitory effects on glycolysis and steroid output; replacement with alanine virtually abolished both these responses. Replacement of arginine in position 8 with lysine in the Phe9 analogue caused a fifty-fold increase in glycolytic potency, but rendered it steroidogenically inactive. Cyclic AMP production was abolished in the Ala9 analogue and greatly impaired in the Phe9 and Lys8,Phe9 analogues. Replacement of the glycine moiety in position 10 with L-alanine, D-alanine, beta-alanine or alpha-aminoisobutyric acid had little or no effect on steroidogenic or glycolytic capacity, although potency was reduced with all substitutions excepting L-alanine.(ABSTRACT TRUNCATED AT 250 WORDS)