Myristoylated alanine-rich C-kinase substrate (MARCKS), a prominent substrate for conventional and novel protein kinase C (PKC) isoforms, is involved in the regulation of membrane–cytoskeletal interactions. Addition of [γ- 32P]ATP to the membrane fraction of digitonin-permeabilized C6 glioma cells resulted in phosphorylation and release of MARCKS, indicating involvement of an active membrane-bound kinase. Pretreatment of cells with 2 μM 4β-12- O-tetradecanoyl-phorbol-13-acetate (β-TPA) for 18 h downregulated conventional (PKCα) and novel (PKCδ) isoforms of PKC by >90% in both membrane and soluble fractions, but did not inhibit the rate of ATP-dependent phosphorylation or release of MARCKS, or decrease levels of membrane-bound PKCζ or PKCμ. MARCKS phosphorylation was inhibited by staurosporine, bis-indolylmaleimide (a PKC-specific inhibitor), Gö6983 (inhibits all isoforms except PKCμ), and a peptide from the calmodulin-binding domain of MARCKS, but was unaffected by EGTA or Gö6976 (inhibits cPKCs and PKCμ). Peptide mapping indicated similar in vivo and in vitro phosphorylation at serine residue(s) known to be phosphorylated by PKC. These findings support a novel mechanism by which MARCKS may be regulated by an atypical PKC isoform in phorbol ester-downregulated cells.