HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.