The myristoylated alanine‐rich C‐kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC β1, novel (n)PKC δ and nPKC ɛ efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC δ (Km= 0.06 μM) was higher than that of nPKC ɛ and cPKC β1 (Km= 0.32 μM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC ɛ > cPKC β1 > nPKC δ. Atypical (a)PKC ζ did not phosphorylate the intact MARCKS protein. However, a 25‐amino‐acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC ζ as well as by the other three PKC.Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.