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Phosphorylation regulates transcription factor activity by influencing dimerization, cellular localization, activation potential, and/or DNA binding. Nevertheless, precisely how this post-translation modification mediates these processes is poorly understood. Here, we examined the role of phosphorylation on the DNA-binding properties of MafA and MafB, closely related transcriptional activators of the basic-leucine zipper (b-Zip) family associated with cell differentiation and oncogenesis. Many common phosphorylation sites were identified by mass spectrometry. However, dephosphorylation only precluded the detection of MafA dimers and consequently dramatically reduced DNA-binding ability. Analysis of MafA/B chimeras revealed that sensitivity to the phosphorylation status of MafA was imparted by sequences spanning the C-terminal dimerization region (amino acids (aa) 279-359), whereas the homologous MafB region (aa 257-323) conveyed phosphorylation-independent DNA binding. Mutational analysis showed that formation of MafA dimers capable of DNA binding required phosphorylation within the distinct N-terminal transactivation domain (aa 1-72) and not the C-terminal b-Zip region. These results demonstrate a novel relationship between the phosphoamino acid-rich transactivation and b-Zip domains in controlling MafA DNA-binding activity.