Actinomycin D caused the irreversible loss of PBS1 phage infectious centers and PBS1-mediated transductants. The loss of infectious centers occurred only within the first 4 min after the addition of phage to cells. Actinomycin did not inactivate free phage or inhibit phage adsorption. Electron micrographs indicated that phage adsorbed to cells in the presence of actinomycin ejected their deoxyribonucleic acid (DNA) normally. However, when cells were infected in the presence of actinomycin, 15 to 22% of their 32P-labeled DNA appeared in the medium, whereas only 1.5 to 7.2% of the 32P-labeled DNA appeared in the medium during normal infection. Neither 8-azaguanine nor chloramphenicol caused a similar loss of PBS1 infectious centers or transductants. Actinomycin also caused the loss of SP10 infectious centers but it had no effect on SP01 or φ29 infections. We conclude that actinomycin causes abortion of PBS1 infection by inhibiting the uptake or retention of phage DNA into host cells. The immunity of SP01 and φ29 infections to actinomycin probably reflects differences in the penetration mechanisms of these phages.