Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.