The expression of intracellular antibodies (intrabodies) in eukaryotic cells has provided a powerful tool to manipulate microbial and cellular signaling pathways in a highly precise manner. However, there have been several technical issues that have restricted their more widespread use. In particular, single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare sFvs from natural libraries or libraries of engineering sFvs that could fold properly in the absence of disulfide bonds were used. Here, we investigated whether target specific sFvs that are isolated from a 15 billion member non-immune human sFv-phage display library could be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity within pools of antigen-specific sFvs. As the target, we used a synthetic transformation effector site 1 (TES1) polypeptide comprising the membrane-most proximal 34 amino acid residues of the carboxy-terminal cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus, which serves as a docking site for adapter proteins of the tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family. Anti-TES1 sFvs, initially identified by phage ELISA screens, were grouped into pools according to the absorbance reading of the antigen-specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors and expressed as cytoplasmic intrabodies. Using the pooling strategy, there was no loss of individual anti-TES1 sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allowed the segregation of individual anti-TES1 sFvs into discrete or minimally overlapping intrabody pools. Further assessment of the biological activity of the anti-TES1 intrabody pools demonstrated that they were all able to selectively block F-LMP1-induced NFkappaB activity that was mediated through the TES1-site and to bind LMP1 protein with high efficiency. This direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.