In the guinea pig adrenal the cells of the outer zone secrete high levels of steroid and respond to ACTH with increased synthesis of cholesterol and steroid. The outer zone consists of two cell types: zona fasciculata (ZF) and zona glomerulosa (ZG). To determine the relative contribution of ZF and ZG to the outer zone's ACTH response, purified populations of each cell type were prepared from ACTH-treated and control animals. Levels of proteins potentially involved in the ACTH response were measured by ELISA. HMG CoA reductase, the rate limiting enzyme of cholesterol synthesis, and two cytochrome P450s, P450(17 alpha) and P450(21), were studied. P450(17 alpha) is required for production of cortisol, but not for corticosterone or aldosterone. P450(21) is required for production of all of these corticosteroids. ZF cells had 4-5 fold greater concentration of all three proteins, but the proteins in ZG cells showed a greater response to ACTH (approximately 3 fold). The response of ZG cells to ACTH and their possession of P450(17 alpha) is consistent with observations made in vitro that ZG cell populations synthesize cortisol and respond to ACTH with increased output of cortisol as well as of corticosterone and aldosterone. In ZG cells to a greater extent than ZF cells, the response to ACTH involved increases in levels of enzymes of both cholesterol and steroid synthesis, suggesting that new protein synthesis is an important component of their ACTH response. On the other hand, the fact that ZF cells can increase steroid output in response to ACTH with a lesser increase in these proteins suggests a different mechanism of regulation. Mobilization of stored cholesterol may be more important in the ACTH-responsiveness of the lipid-filled ZF cells than in the lipid-poor ZG cells.