We have examined cytokeratins from stratum corneum (s. corneum) of the palm and back of hands by biochemical techniques which characterize cytokeratin No. 9 (CK9). By two-dimensional gel electrophoresis, cytokeratins from s. corneum of the palm (PSC) were resolved into three spots (designated as CK-A, CK-B and CK-C by us), and cytokeratins from s. corneum of the back of hands (BSC) were resolved into two spots (CK-A and CK-C). The results of polypeptide mapping and the spot positions in two-dimensional gels suggest that CK-A, CK-B and CK-C are degraded products derived from CK1/2, CK9 and CK10/11 in living keratinocytes of epidermis, respectively. While the spots of CK-A and CK-B were prominent and the spot of CK-C was very weak in PSC, the spots of CK-A and CK-C were prominent and the spot of CK-B was not detected in BSC. In filament reconstitution in vitro, cytokeratins from PSC formed intermediate-sized filaments similar to those commonly observed using cytokeratins from whole epidermis. In contrast, cytokeratins from BSC formed only short filaments and small granules. In order to examine this difference in detail, polypeptides were eluted electrophoretically from gel slices containing spots. Addition of eluted CK-B to cytokeratins from BSC promoted the formation of intermediate-sized filaments similar to those from PSC. Furthermore, although the mixture of eluted CK-A and CK-C did not form intermediate-sized filaments, the combination of CK-A and CK-B alone did form them.(ABSTRACT TRUNCATED AT 250 WORDS)