Treatment of tumor cells with hydroxyurea (HU) has been shown to increase the experimental metastatic potential of these cells. We have previously described the induction of stress proteins (antioxidants) by HU in B16 murine melanoma cells and their relationship to the metastatic process. We have now investigated the induction by HU of another set of stress proteins, the heat shock proteins, and their role in experimental metastasis. HU markedly increased the cellular content of heat shock protein (hsp) 27 but not of hsp 90, 72/73, or 60 as measured by immunoblotting. The induction of hsp27 protein was preceded by a specific increase in hsp27 mRNA. Furthermore, HU-treated cells were more thermotolerant. To investigate the functional role of hsp27, human hsp27 cDNA was constitutively overexpressed in B16 cells at levels seen in HU-treated cells. In separate experiments, we induced a global increase in hsps by heat shock. Neither the hsp27 transfectants nor the heat-shocked cells demonstrated an increase in their experimental metastatic capacity. We conclude that hsp27 protein is increased by HU by the specific induction of hsp27 mRNA in B16 melanoma cells but increased hsp27 protein is not responsible for the increase in experimental metastasis. Since high levels of hsp27 are associated with metastatic disease in breast and ovarian cancers, but not in our experimental system, the functional role of hsp27 in metastasis requires further study.