Abstract ACTH-induced increases in adrenal polyphosphoinositides were demonstrable after extraction by acid or non-acid methods, and after purification by unidimensional or two-dimensional thin layer chromatography. ACTH-induced increases in phosphatidylinositol mass were apparent, both as increases in measurable phosphorus and inositol. ACTH and insulin also increased 32PO4 incorporation into adrenal and adipose tissue polyphosphoinositides which were acid-extracted and purified by two-dimensional chromatography. Cyclic-AMP increased the mass of phosphatidylinositol, but decreased 32PO4 incorporation into this and other phospholipids; the cause for this decrease in specific activity was not evident. Increasing doses of cycloheximide progressively inhibited ACTH- or insulin-induced increases in the mass of phosphatidylinositol, but 32PO4 incorporation therein followed a bimodal curve, with inhibition at lower doses and stimulation at higher doses; these divergent changes in phosphatidylinositol mass and 32P-labeling at higher concentrations raise the possibility that cycloheximide may activate phospholipases in hormone-stimulated tissues. These results offer further support for our contention that ACTH and insulin increase inositide phospholipid concentrations in their target tissues by a cycloheximide-sensitive mechanism.