During hair follicle differentiation, several families of keratin proteins are synthesized sequentially. In the present study, cDNA clones encoding six members of mouse high-glycine/tyrosine protein were isolated by screening a cDNA library prepared from the mouse skin of an anagen phase with a differential hybridization technique. On the basis of their nucleotide and deduced amino acid sequences, they were found to encode two members of high-glycine/tyrosine protein type I and four of type II. Interestingly, one of the four type II proteins had been encoded by two distinct cDNAs. Among the cDNA clones isolated were included the ones encoding a new member of type I and II protein, respectively, which possessed an entire open reading frame. Novel type II protein, termed type II.4, with a molecular mass of 15,130 Da was revealed to have significant direct repeats and a cysteine residue at the carboxyl terminus, which indicates that this protein has characteristics intermediate between high-glycine/tyrosine proteins and cysteine-rich proteins. In addition, the new member of type I protein has some features common with type II protein. We propose to term this protein type Ialpha until it is further characterized. Northern blot analysis demonstrated that gene expression of mouse high-glycine/tyrosine proteins followed the hair cycle growth fundamentally and reached its peak at day 9 in the first hair cycle, while two peaks of their expression were observed at day 33 and day 39 in the second cycle. Their transcripts were expressed in the cortical cells of hair follicles but not in the cells of the outer root sheath, inner root sheath, or medulla. Moreover, their gene expression commenced at different levels in cortical cells. The novel findings that each gene is activated transcriptionally with a distinct expression pattern spatially and temporally suggest that there is a remarkable difference in the distribution of these proteins in hair.