3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis, was examined in the lipid-filled outer cortical cells and smooth endoplasmic reticulum-filled inner cortical cells of guinea pig adrenals. The specific activity of HMG CoA reductase was higher in microsomes obtained from the outer cortex, but was stimulated by ACTH and suppressed by dexamethasone (DEX) in both regions. Immunoblots of the microsomal proteins revealed a higher concentration of immunoreactive HMG CoA reductase (mol wt 94K) in microsomes from outer cortical cells. Changes in the intensity of this band corresponded to changes in activity after treatment with ACTH and DEX. However, quantitative immunoassay revealed that changes in activity in the inner zone after ACTH and in both zones after DEX were greater than could be accounted for by changes in immunodetectable protein, implying that other regulatory factors play a role in these cases. Incubation of outer and inner cortical tissues in vitro showed that outer cortical tissue had a greater capacity to incorporate [14C]acetate into cellular cholesterol (free and esterified) and into secreted steroid than did inner cortical tissue. ACTH enhanced the incorporation of [14C]acetate by outer cortical tissues into secreted steroid, but had little effect on incorporation by inner cortical tissues. Assay of the medium indicated that outer cortical tissues secreted much more steroid and increased secretion in response to ACTH, whereas inner cortical tissues secreted little steroid and were little affected by ACTH. Taken together, these results show that outer cortical cells have a greater capacity for cholesterol synthesis and that enhancement of this capacity after ACTH treatment is correlated with an increase in HMG CoA reductase protein. On the other hand, while the level of HMG CoA reductase immunodetectable protein and activity in inner cortical cell microsomes is considerable and appears to increase after ACTH treatment, it is not translated into an ability to synthesize cholesterol. This suggests that the activity of the immunodetectable HMG CoA reductase in the inner zone is suppressed in vivo and that the prominent smooth endoplasmic reticulum found in inner cortical cells has additional functions. The inability of the inner zone to synthesize cholesterol accounts in part for its low steroid production.