Caspase-14 is expressed in a tissue-specific manner in mouse skin. Hero we determined the complete caspase-14 cDNA sequence of human caspase-14 by rapid amplification of cDNA ends. Sequence comparison with a cosmid clone containing genomic DNA revealed that the human caspase-14 gene comprises seven exons. Facultative utilization of a cryptic splice acceptor site within intron 5 leads to the formation of two mRNA species. In situ hybridization of human skin showed that caspase-14 is expressed in the uppermost layer of living epidermal cells, i.e., the granular layer, in hair follicles and sebaceous glands, In vitro caspase-14 transcription was low in subconfluent cultures but strongly increased when keratinocyte differentiation was simulated by maintaining cells at confluence for several days. This transcriptional upregulation was suppressed in the presence of a high extracellular calcium concentration. Our findings show that caspase-14 is regulated at the level of transcription during keratinocyte differentiation in vitro and in vivo.