Preparations of cytoskeleton from Y-1 cells were found to phosphorylate various cytoskeletal proteins when incubated with [gamma-32P]ATP. When cAMP was added to the cytoskeleton, a rapid increase in phosphorylation of cytoskeletal protein was observed, and changes were seen in the phosphorylation of individual proteins; four additional proteins were phosphorylated (mol wt, 165,000, 92,000, 45,000, and 24,000) and three proteins were more intensely phosphorylated than without cAMP (mol wt, 125,000, 51,000, and 38,000). In addition, one protein (mol wt, 96,000) that was intensely phosphorylated without cAMP was not phosphorylated with the cyclic nucleotide, and a second (mol wt, 48,000) was less phosphorylated. The increased level of total phosphorylation returned to the unstimulated level within 10 min. The increased phosphorylation of proteins produced by cAMP was inhibited by protein kinase inhibitor. cAMP-dependent protein kinase activity was closely associated with the cytoskeleton, since it was not removed by Triton X-100 (1%, wt/vol), although some activity could be extracted with buffer containing high concentrations of salt. When the cytoskeleton of Y-1 cells was subjected to treatments that disrupt the cytoskeleton before the cells were extracted (cytochalasin B, colchicine, and sonication), no change was seen in cAMP-dependent protein kinase activity. However, cytochalasin B increased phosphorylation of two proteins that were not phosphorylated by cAMP-dependent kinase (mol wt, 63,000 and 43,000). Sonication of the cytoskeleton before addition of [gamma-32P]ATP caused a number of changes in cAMP-independent phosphorylation, but did not affect cAMP-dependent phosphorylation. cAMP-dependent phosphorylation required Mg2+ and was inhibited by Ca2+. It is concluded that the cytoskeleton of Y-1 cells contains bound cAMP-dependent protein kinase that phosphorylates certain cytoskeleton proteins. The cytoskeleton also contains one or more cAMP-independent kinase systems. It is suggested that the cAMP-dependent protein kinase described here may be important in the cytoskeletal responses to ACTH.