The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG‐01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG‐01 expressed mRNAs of PKCα, ‐βI, ‐βII, ‐δ, ‐ε, ‐η, ‐θ and ‐ζ, but not PKCγ. At the protein molecule level, MEG‐01 was observed to express PKCα, ‐βI, ‐βII, ‐ε, ‐θ and ‐ζ, but lack ‐γ, ‐δ and ‐η. When differentiation of MEG‐01 was induced by 100 nM 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), rapid translocation from cytosol to membrane fraction and down‐regulation of PKCα, ‐ε and ‐θ was observed in 1–2 h. On the other hand, PKCβI and ‐βII were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down‐regulation, especially βII, was very slow. The myristoylated, alanine‐rich C kinase substrate (MARCKS) in the membrane fraction of MEG‐01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time‐course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKCα, ‐ε and ‐θ is involved in the initiation of differentiation, and that PKCβI and ‐βII have important roles in the maintenance of differentiation. Although PKCζ was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.