Signaling proteins often contain multiple modular protein-protein interaction domains of the same type. The Saccharomyces cerevisiae checkpoint kinase Rad53 contains two phosphothreonine-binding forkhead-associated (FHA) domains. To investigate if the precise position of these domains relative to each other is important, we created three rad53 alleles in which FHA1 and FHA2 domains were individually or simultaneously transposed to the opposite location. All three mutants were approximately 100-fold hypersensitive to DNA lesions whose survival requires intact Rad53 FHA domain functions, but they were not hypersensitive to DNA damage that is addressed in an FHA domain-independent manner. FHA domain-transposed Rad53 could still be recruited for activation by upstream kinases but then failed to autophosphorylate and activate FHA domain-dependent downstream functions. The results indicate that precise FHA domain positions are important for their roles in Rad53, possibly via regulation of the topology of oligomeric Rad53 signaling complexes.