In previous studies, an enhanced expression with abnormal phosphorylation of a low molecular weight heat shock protein, HSP27, was identified in reactive astrocytes and Rosenthal fibers in Alexander disease brain. To investigate the relevance of HSP27 to the pathological change of astrocytes, phosphorylation of HSP27 was examined in astrocyte cultures isolated from fetal human brains. In unstimulated human astrocytes, HSP27 was identified in the cytoplasm and was comprised of a major unphosphorylated "a" isoform and a minor monophosphorylated "b" isoform. The level of HSP27 phosphorylation was elevated greatly after a 30 minute exposure to heat shock, sodium arsenite, interleukin-1 (IL-1 alpha and IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) with an increased expression of a diphosphorylated "c" isoform. Treatment with interferon-beta (IFN-beta), platelet-derived growth factor-AA, leukemia inhibitory factor, phorbol 12-myristate 13-acetate, and dibutyryl cyclic AMP stimulated phosphorylation of HSP27 moderately, while IFN-gamma, TNF-beta, basic fibroblast growth factor, epidermal growth factor, or fetal bovine serum did not significantly alter the level of HSP27 phosphorylation. Total amount of the HSP27 protein and its cytoplasmic localization were unaffected by any of these reagents. These results indicate that HSP27 is a constitutive protein in human astrocytes. The induction of HSP27 phosphorylation by a specific set of cytokines and growth factors suggests that HSP27 is a key cellular substrate by which signaling events are mediated in human astrocytes under normal and pathological conditions.