We have identified in mammalian cells a novel cyclic AMP response element (CRE)-binding protein of molecular mass 47 kDa. This protein was not recognized by either the CREB-327/341 or c-Jun antisera, and its tissue distribution did not overlap with those of the CREB and Jun families. For example, hepatoma and placental tissue did not contain the 47-kDa DNA-binding protein, but did contain the CREB isoforms. On the other hand, S49 lymphoma cells contained a high level of the 47-kDa DNA-binding protein but did not contain a 47-kDa Jun-related protein which was found in normal liver and hepatoma. This new 47-kDa factor bound to the CRE in the dephosphorylated form, and phosphorylation of the protein by the catalytic subunit of protein kinase A completely abolished its DNA-binding activity. The isoforms of the CREB-327/341 family, on the other hand, bound to DNA in the phosphorylated form, and alkaline phosphatase treatment reduced significantly their interaction with CRE sequence. This reverse effect of phosphorylation/dephosphorylation on the DNA-binding property of this new 47-kDa protein in particular distinguishes it from other known CREB factors and suggests that the protein might play a unique role in the regulation of cAMP-mediated transcription.