The Janus Kinases (JAK) JAK1, JAK2, and TYK2 are protein tyrosine kinases which play a pivotal role in the signal transduction process mediated by cytokines. These kinases appear to transduce signals via their substrates which modulate programs of gene expression specific to the respective signals. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (GCSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. To determine if additional members of JAK kinase family exist, we designed a polymerase chain reaction based strategy which resulted in the identification of a new member of the JAK kinase family. This new kinase, which we have named JAK3 is encoded by a 4.3 kb mRNA transcript. Nucleotide sequence analysis of a full length cDNA derived from this mRNA revealed that it encodes an open reading frame of 3897 bp. The protein encoded by this mRNA contains the double catalytic domain characteristic of the JAK family kinases. The most striking difference between JAK3 and the other JAK kinases is the presence of two stretches of additional amino acid sequence of 147 and 28 residues which span between amino acid positions 322 to 469 and 632 to 660 respectively. Expression studies indicate that JAK3 is expressed at very low levels in immature hematopoietic cells, but its expression is dramatically up-regulated during terminal differentiation of these cells. These results suggest that JAK3 plays an important role in the differentiation of hematopoietic cells.