S100B and S100A6 (calcylin) are two members of the S100 Ca(2+)-binding protein family and have been localized in the mammalian nervous system. However, information on their distribution in the human nervous system, especially in the developing human fetal brain, is scarce. In the present study, an immunocytochemical method was used to examine the spatio-temporal protein expression patterns of S100B and S100A6 in normal human fetal hippocampus, entorhinal cortex and occipital cortex. Normal aged adult human brain specimens were also included for comparison. From week 15 onwards, an increase with advancing gestation age in both the number and staining intensity of S100B positive, astrocyte-like cells was found in the pyramidal layer of the hippocampus, while both the molecular and polymorphic layers showed similar S100B immunoreactivities at all stages examined. A decrease in the immunoreactivities was found in the molecular layer of the aged adult hippocampus while other layers exhibited immunoreactivities similar to those of the late fetus. At week 15, the molecular, pyramidal and ganglionic/multiform layers of the entorhinal cortex also showed positive S100B immunoreactivities which were maintained throughout the rest of the gestation and in adult specimens. In the occipital cortex, the numbers of positive cells for all layers were about twofold higher than those found in the hippocampus and entorhinal cortex, and immunoreactivities detected in the granular layer increased from week 21, reaching a plateau at around week 27. S100B positive fibers were also found at week 30 but were not observed in aged adult specimens. S100A6 positive cells were on the whole fewer in number than those of S100B in the brain regions examined. The S100A6 immunoreactivities which were localized in some pyramidal neuron-like and some glial-like cells of the pyramidal and molecular layers of the hippocampus increased by midgestation and became weak in the late fetus and in aged adult specimens. Weakly stained S100A6 positive cells were also observed in the entorhinal cortex throughout the gestation and in aged adult cortex. S100A6 immunoreactivities were weak in the fetal occipital cortex. They were also localized in the glial-like cells of the aged adult occipital cortex. The differential spatio-temporal expression of S100B and S100A6 proteins suggests that the proteins play different roles in different brain regions during development and in adulthood.