We have previously demonstrated that human cells contain multiple forms of uracil-DNA glycosylase (Caradonna, S. J., Ladner, R., Hansbury, M., Kosciuk, M., Lynch, F., and Muller, S. J. (1996) Exp. Cell Res. 222, 345-359). One of these is an Mr 29,000 processed form of the highly conserved uracil-DNA glycosylase (UDG1) located in the mitochondria. The others are located in the nucleus and migrate as a group of at least three distinct bands within the 35,000-37,000 molecular weight range. In this report, we perform a detailed characterization of the Mr 35,000-37,000 purified proteins. To accomplish this, uracil-DNA glycosylases were affinity purified from HeLa cell nuclear extracts. The proteins were separated by SDS-PAGE, and their identities were verified by renaturation and activity assays. The three protein bands were individually digested with cyanogen bromide, and the resulting peptide fragments were analyzed by direct amino acid sequencing. Peptide sequence, derived from each band, was identical and corresponded to a recently identified isoform of UDG1. This isoform (UDG1A) has a unique 44-amino acid N-terminal region and a C-terminal region that is identical to UDG1. To begin to study the signals required for nuclear _targeting, the N-terminal regions of UDG1 and UDG1A were isolated and cloned into pEGFP-N2 to generate fusions with a red-shifted variant of green fluorescent protein (GFP). When these constructs were transfected into NIH3T3 cells, UDG1/pEGFP was _targeted to the mitochondria, and UDG1A/pEGFP was _targeted to the nucleus. Further studies, using deletion mutants, demonstrate that the nuclear localization signal resides within the first 20 amino acids of UDG1A. To investigate the possibility that the heterogeneity observed on SDS-PAGE results from post-translational modification(s), the UDG/pEGFP fusion constructs were transfected into NIH3T3 cells, and the cells were metabolically labeled with [32P]orthophosphate. Results from these experiments show that UDG1A is a phosphoprotein. Subsequent phosphoamino acid analysis revealed that UDG1A is phosphorylated on both serine and threonine residues. As a final characterization, RNase protection assays were performed to examine expression of each of these isoforms. These studies demonstrate that UDG1A is expressed in a wide variety of cell types and that message levels are elevated in transformed cells.