Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant γ1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells curry a defect in, and can also be complemented by, the β- subunit of the IFN-γ receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant γ2A cells lack detectable JAK2 mRNA and protein. By using γ2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN- γ signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-γ. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-γR2 subunit requires the amino-terminal region but nut the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-α and -γ in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.