Loricrin, involucrin, small proline-rich protein (SPRR)1, SPRR2, and SPRR3 genes are located within a cluster of 1.5 Mbp on chromosome 1q21 and most likely evolved from a common ancestor. Monospecific polyclonal antibodies and cDNA probes were produced to investigate SPRR transcripts and proteins. SPRR expression was restricted to terminally differentiating squamous cells, preferentially located at the cell periphery, and immunoreactivity was greatly reduced in cells with a mature cornified cell envelope. Furthermore, detectable SPRR2 and SPRR3 levels were strongly increased in differentiating keratinocyte cultures after addition of LTB-2, a specific inhibitor of transglutaminases, suggesting that they are precursor proteins of the cornified cell envelope. In normal epidermis, SPRR1 was restricted to appendageal areas, SPRR2 was expressed coherently, and SPRR3 was completely absent. In the upper digestive tract, SPRR1 was expressed in sublingual and tongue epithelium, SPRR2 was mostly restricted to lingual papillae, and SPRR3 was abundant in oral and esophageal epithelium. In psoriatic epidermis, SPRR1 and SPRR2 were expressed at much higher levels than in normal epidermis. Addition of 10-7 M retinoic acid to cultured differentiating keratinocytes significantly down-regulated the expression of SPRR2 and SPRR3 transcripts and slightly decreased that of SPRR1. Thus, SPRR1, SPRR2, and SPRR3 are differentially expressed in vivo and in vitro, suggesting that the SPRR multigene family evolved to serve as highly specialized cornified cell envelope precursor proteins in stratified epithelia.