A cDNA clone encoding human epidermal keratin No. 5 (K5) has been isolated from a lambda gt10 cDNA library on the basis of crosshybridization with cDNAs coding for other basic keratins and differential hybridization with total cDNA probes. This clone contains a 1100-bp insert able to inhibit specifically translation of K5 in a hybrid-arrested translation test. Northern blots show that this insert hybridizes with a 2.4-kb message present in epidermal mRNAs. The 1100-bp sequence reported here corresponds to the 3' half of the K5 message. Comparison of the deduced aminoacid sequence shows that the protein exhibits characteristic features of a basic keratin. The 242 aminoacid sequence reported here extends from the carboxyterminal end up to the last helical portion of the central rod domain.