Bacteriophage T4 makes a large number of prereplicative proteins, which are involved in directing the transition from host to phage functions, in producing the new T4 DNA, and in regulating transcriptional shifts. We have used two-dimensional gel electrophoresis (nonequilibrium pH gradient electrophoresis gels in the first dimension and sodium dodecyl sulfate-polyacrylamide gradient slab gels in the second) to identify a number of new prereplicative proteins. The (18), who used one-dimensional gels. MATERIALS AND METHODSPhage and bacterial strains. The phage strains used are listed in Table 1. amber mutant strains were grown from single plaques on E. coli K803 (rK-mK-thi met supE). Other strains were usually grown on E. coli B or B834 (rB-mB-thi met), which were also used for labeling experiments.