The bovine herpesvirus 1 (BHV-1) U_L3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U_L3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U_L3.5 is deficient in viral egress but can be complemented by BHV-1 U_L3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). The function of BHV-1 U_L3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U_L3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 -transinducing factor (BTIF) as a BHV-1 U_L3.5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U_L3.5 and BTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U_L3.5, but not 40 amino acids from the C terminus, abolished the U_L3.5-BTIF interaction both in vitro and in vivo. The interaction between U_L3.5 and BTIF may be important for BHV-1 maturation and regulation of BTIF transactivation activity.