The M(r) 27,000 heat shock protein (hsp27) is a member of the small heat shock protein family. Cell differentiation is a process in which a role for small heat shock proteins has been suggested. The ability to control the state of differentiation in normal human keratinocytes by modification of extracellular calcium concentration makes it an ideal in vitro system for exploration of the specific components and steps in differentiation. We have focused on the in vitro expression of hsp27 in undifferentiated and differentiated human normal keratinocytes (HNK) as a marker for differentiation. Immunological methods (immunohistochemistry and immunoblotting) as well as Northern blotting were used. Cells of the breast cancer line MCF-7 served as a positive control. We demonstrated that hsp27 was expressed at low levels in normal human keratinocytes, kept under calcium concentrations where cells formed discrete colonies of undifferentiated, noncornified cuboidal cells (0.03 mM Ca2+), and linked cuboidal cells with a noncornified appearance (0.15 mM Ca2+). Upon cultivation in high calcium (1.00 mM Ca2+) where a more morphological state of differentiation was reached, more spindle shaped with cornification of individual cells, a 2-fold increase in hsp27 expression was observed. A somewhat weaker increase in hsp27 mRNA was shown by Northern blot analysis. Our studies provide evidence that hsp27 is accumulated in a differentiation-dependent manner in human normal keratinocytes grown under conditions inducing terminal differentiation (0.03-1.00 mM Ca2+). Therefore, hsp27 can be regarded as a marker of differentiation in human normal keratinocytes.