We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.