Protein synthesis by bovine adrenal glomerulosa and fasciculata cells in response to various modulators of steroid synthesis was examined using [35S]methionine labeling and two-dimensional gel electrophoresis. Both cell types responded to steroidogenic stimuli with rapid changes in a family of 28- to 30-kilodalton (kDa) proteins similar to those described in rat fasciculata by Epstein and Orme-Johnson. In glomerulosa, angiotensin-II (AII), potassium, and (Bu)2cAMP stimulated the appearance of two 28.5-kDa proteins (no. 3 and 4) with pI values of 6.44 and 6.33 and decreased labeling of two other 28.5-kDa proteins (no. 1 and 2) with pI values of 6.9 and 6.59. The rank order of potency on aldosterone synthesis and that on proteins 1-4 were the same: (Bu)2cAMP > AII > potassium. Atrial natriuretic peptide blocked the effects of AII on all four proteins and on aldosterone synthesis. Adrenal secretagogues also affected labeling of four slightly larger (30 kDa) proteins (no. 5-8). Corresponding proteins in each quartet are separated by the same difference in isoelectric points. These eight proteins may represent a core protein systematically modified in a number of ways. Aldosterone synthesis in glomerulosa, like glucocorticoid synthesis in fasciculata, requires ongoing protein synthesis. The 28- to 30-kDa proteins increased by steroidogenic stimuli in both cells and decreased by atrial natriuretic peptide in glomerulosa may be the proteins whose synthesis is crucial to acute control of steroidogenesis. Our results indicate that these proteins are made in response to calcium- or calcium/phosphoinositide-dependent mechanisms as well as by cAMP.