In the human keratinocyte line HaCaT, the nonphosphorylated 27-kDa heat shock protein (HSP27) isoform A (pI 6.5) is constitutively expressed. Application of thapsigargin, which inhibits Ca2+-ATPase in the endoplasmic reticulum, results in the rapid formation of the phosphorylated HSP27 isoform B (pI 6.0) and reduction of HSP27 A without affecting the synthesis of HSP27. The thapsigargin-dependent HSP27 isoform change is similar to that induced by 43 degrees C heat shock, but different from that induced by arsenite, where the biphosphorylated isoform HSP27 C (pI 5.7) is observed. The receptor agonist bradykinin, which increases intracellular Ca2+ (Ca(i)) level, shows no effect on the distribution of HSP27 isoforms. The responses of HSP27 isoforms to thapsigargin are independent of Ca(i) concentration in HaCaT cells. These observations suggest that the thapsigargin-induced change in HSP27 isoforms is dependent on the depletion of internal Ca2+ stores rather than on the increase in Ca(i) concentration. The thapsigargin-induced change in HSP27 isoforms is reduced by the tyrosine kinase inhibitor, genistein, but not the protein kinase C inhibitor, H-7. We propose that the modulation of HSP27 phosphorylation status by Ca(i) homeostasis may be mechanistically linked to control of keratinocyte growth and differentiation and responses of keratinocytes to extracellular stresses.