This study describes effects of ACTH1–24 and β-endorphin on brain polyphosphoinositide metabolism in vitro. The interconversion of these polyanionic phospholipids was studied by incubation of a lysed synaptosomal fraction with [γ-32P]ATP. Of the membrane phospholipids only PA, DPI and TPI became labeled. The reference peptide ACTH1–24 stimulated the formation of TPI and inhibited the production of PA. For effects on TPI formation both the sequences ACTH5–7 and ACTH10–16 were needed. Effects of PA formation required the sequences ACTH7–10 and ACTH10–16. The basic amino acids in ACTH10–16 seemed to be of crucial importance for the peptide effects. A stimulatory effect on DPI was visible when ACTH was shortened from the N-terminus, and the essential information was in ACTH7–10. β-endorphin inhibited PA formation and this effect was abolished by C-terminal shortening to γ-endorphin. Other fragments of the C-terminus of β-LPH, including the enkephalins, were ineffective. It is concluded that the structure-activity relationship obtTPI/PA formation correlates with a similar relationship obtained on excessive grooming behavior in vivo. A possible correlation between the effects on polyPI metabolism and opiate-like effects, and effects on extinction of active avoidance behavior in vivo is discussed.