The packaging proteins, gp2 and gp3, of Salmonella phage P22 were separated by DEAE-sephacel column chromatography. The activity of both proteins was measured by complementation in an in vitro phage packaging assay. For the first time gp3 activity was demonstrated in vitro. The majority of the gp3 activity could be separated from gp2. While gp2 was stable during chromatography and storage, gp3 was very unstable. However, it was stable when stored as a crude extract. The ratio and interaction of these two proteins critical for phage maturation are still uncertain.