Polymerase chain reaction-based screening of an arrayed human P1 artificial chromosome (PAC) library using primer pairs specific for the human type I hair keratins hHa3-II or hHa6, led to the isolation of two PAC clones, which covered 190 kilobase pairs (kbp) of genomic DNA and contained nine human type I hair keratin genes, one transcribed hair keratin pseudogene, as well as one orphan exon. The hair keratin genes are 4-7 kbp in size, exhibit intergenic distances of 5-8 kbp, and display the same direction of transcription. With one exception, all hair keratin genes are organized into 7 exons and 6 positionally conserved introns. On the basis of sequence homologies, the genes can be grouped into three subclusters of tandemly arranged genes. One subcluster harbors the highly related genes hHa1, hHa3-I, hHa3-II, and hHa4. A second subcluster of highly related genes comprises the novel genes hHa7 and hHa8, as well as pseudogene PsihHaA, while the structurally less related genes hHa6, hHa5, and hHa2 are constituents of the third subcluster. As shown by reverse transcription-polymerase chain reaction, all hair keratin genes, including the pseudogene, are expressed in the human hair follicle. The transcribed pseudogene PsihHaA contains a premature stop codon in exon 4 and exhibits aberrant pre-mRNA splicing. Evolutionary tree construction reveals an early divergence of hair keratin genes from cytokeratin genes, followed by the segregation of the genes into the three subclusters. We suspect that the 190-kbp domain contains the entire complement of human type I hair keratin genes.