The adenovirus 33K protein appears to be a multifunctional protein performing different roles in viral infection. The involvement of 33K protein in different steps of adenovirus replication may require protein-protein interaction. Using 33K protein as a bait in the yeast two-hybrid system, we screened open reading frames (ORFs) of bovine adenovirus (BAdV)-3 for potential interactions with 33K protein. Interestingly, 33K protein showed specific interaction with 100K and pV proteins. The yeast two-hybrid findings were validated by in vitro binding using in vitro synthesized transcription-translation products, GST pull down assay and in vitro co-immunoprecipitation assay using protein-specific antibodies. We demonstrated, that the interaction of 33K protein with 100K and pV proteins takes place during BAdV-3 infection. Finally, our data suggests that the stretch of amino acids 81-120 and 161-200 in 33K protein are critical for the interaction with pV and 100K proteins, respectively.