Down's syndrome (DS) is the most common chromosomal abnormality in live born infants. The cloning and characterization of genes on HC21 are necessary steps for the understanding of the molecular basis of Down's syndrome. To search for new Down's syndrome related genes, we have used bioinformatics analysis and rapid amplification of cDNA ends to identify an alternatively splicing cDNA of human PKNOX1 in Down's syndrome critical region on the long arm of human chromosome 21. The alternatively splicing full length cDNA of human PKNOX1, named PKNOX1B, is 2 793 bp in length and encodes a 405 amino acid protein. Bioinformatical studies show PKNOX1B gene spans over 58 kb on chromosome 21q22.3 and contains 11 exons and 10 introns. The predicted molecule weight of PKNOX1B is 44.628 kDa, and the deduced iso-electric point is 6.28. Compared with PKNOX1 gene, PKNOX1B is 30 aa shorter at the C terminus than PKNOX1 owing to alternative splice between (10th exon and 11th exon) last two exons. PKNOX1B posses a same homeobox-domain as PKNOX1, so the isoform of PKNOX1 may be involved in the genetic control of development like other members of homeobox-containing gene family. RT-PCR results showed that PKNOX1B is expressed in all examined tissues except in marrow tissue. By Northern blot, PKNOX1 has three transcripts in adult testis, one about 5 kb, second about 2.9 kb, and third about 2 kb. The two larger transcript is expressed in all examined tissues, but the smallest transcript is only expressed actively in adult testis, so that it may play potential roles in spermatogenesis.