Infection of Escherichia coli by bacteriophage T4 leads to the expression of three phage mono-ADP-ribosyltransferases (namely, Alt, ModA, and ModB), each of which modifies a distinct group of host proteins. To improve understanding of these interactions and their consequences for the T4 replication cycle, we used high-resolution two-dimensional gel electrophoresis and mass-spectrometry to identify some of the putative target proteins ADP-ribosylated in vitro by Alt (total approximately 27) and ModB (total approximately 8). E. coli trigger factor and the elongation factor EF-Tu were 2 targets of ModB action, and these proteins were among the 10 identified as targets of Alt, hinting that these factors are involved in phage replication.