Members of the low molecular weight heat shock protein (hsp) family show regulated expression in both Drosophila and mice during development and differentiation. Here we have examined whether similar regulation of the single low molecular weight hsp (hsp 28) of humans exhibits differences in either its expression and/or phosphorylation during the course of in vitro differentiation of hematopoietic cells. In the promyelocytic leukemic cell line, HL-60, we show that early after commitment of the cells to a macrophage-like phenotype (via exposure to phorbol ester myristate, PMA) there occurs an accompanying increased phosphorylation of hsp 28. Over time and as the cells become terminally differentiated the levels of hsp 28 increase significantly. In contrast, cells stimulated to adopt a granulocyte-like phenotype (e.g. exposed to either dimethyl sulfoxide or retinoic acid) show no changes in either the phosphorylation or expression of hsp 28. Moreover, once differentiated the granulocyte-like cells no longer appear capable of phosphorylating hsp 28. Human K562 cells, in response to hemin, rapidly increase their expression and phosphorylation of hsp 28 during the course of their differentiation into erythroid-like cells. Addition of PMA to the K562 cells induces differentiation into a megakaryocyte-like phenotype but is not accompanied by changes in hsp 28 phosphorylation/expression. In the case of the HL-60 cells, differentiation toward the macrophage like lineage is accompanied by an increased adherence of the cells to their substratum and an apparent association of hsp 28 with the actin cytoskeleton.