The major DNA-binding protein, or infected-cell protein 8 (ICP8), encoded by herpes simplex virus can localize to the cell nucleus independently of other viral proteins. To define the nuclear localization signals within ICP8, we performed several forms of mutagenesis on the cloned ICP8 gene. Deletion analysis of the ICP8 gene showed that several portions of ICP8 are involved in its nuclear localization. To determine whether these regions were independent localization signals, we introduced various portions of the ICP8 gene into a series of cassette plasmids which allowed expression of fusion proteins containing pyruvate kinase, normally a cytoplasmic protein, fused to various portions of ICP8. These results showed that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of targeting protein kinase into the nucleus. However, inclusion of certain additional regions of ICP8 into the fusion protein led to an inhibition of nuclear localization. Therefore, the carboxyl-terminal 28 residues of ICP8 can act independently as a nuclear localization signal, but certain conformational constraints or folding or assembly requirements in the remainder of the protein can affect the nuclear localization of the protein. Our results demonstrate that sequences distant from a nuclear localization signal can affect its ability to function. A set of fusion vectors has been isolated which should be of general use for making 5' or 3' fusions in any reading frame to rapidly map localization signals.