EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function.