Desmosomes represent a special type of the plaque-bearing adhering junctions, characteristic of certain pathways of cell differentiation, which compositionally are not identical in the various kinds of desmosome-forming cells. While all desmosomes contain the cytoplasmic plaque proteins desmoplakin I and plakoglobin, they can vary in their specific complement of desmosomal cadherins and by the presence of additional plaque proteins. We have raised monoclonal antibodies recognizing one such ‘accessory’ plaque protein, the cytokeratin-binding, basic protein plakophilin 1, originally introduced as ‘band 6 protein’ or ‘polypeptide D6’, which is an abundant desmosomal component in certain epithelia. Using such antibodies, we have isolated cDNA clones encoding the bovine and the human protein and determined their complete amino acid sequences. The mRNAs, which on Northern blot tests appear as two bands corresponding to approximately 4 and 2.4 kb (bovine) or 5 and 2.6 kb (human), code for 727 amino acids (calculated mol. wt. 80,180; IEP 9.25) in bovine and 726 amino acids (mol. wt. 80,496; IEP 9.34) in human plakophilin. Sequence analyses have revealed the presence of 9.2 repeated units of the arm-motif sequence, confirming our previous conclusion that this protein is a member of a larger family of proteins including, inter alia, several membrane-associated plaque proteins such as vertebrate plakoglobin and β-catenin as well as the product of the armadillo gene of Drosophila. The plakophilin antibodies and cDNA probes have also allowed us to examine its synthesis in various tissues and cell cultures. While we confirm the occurrence of the protein in cytoskeletal fractions from various stratified squamous, complex, glandular duct and bladder epithelia, where it can be localized to desmosomes, we have, surprisingly, also identified the protein, although at lower amounts, in cytoskeletal fractions from several cultured cell lines in which the protein has not been consistently localized to desmosomes by immunofluorescence microscopy. Examples include cultured cells derived from certain simple epithelia such as the kidney-derived line MDBK and cultured calf lens cells. We have also found that, in all plakophilin 1-positive cells examined, a pool of diffusible (‘soluble’) cytoplasmic plakophilin exists, including cell lines such as human mammary carcinoma MCF-7 cells in which this soluble plakophilin seems to be the only detectable form. In addition, we have identified some soluble proteins conspicuously cross-reacting with plakophilin 1. Possible functions of plakophilin and its potential value as a marker for specific states of cell differentiation are discussed, particularly with respect to tumor diagnosis.