Two cell lines [R(B77) and XC] of Rous sarcoma virus-transformed rat cells were infected with herpes simplex virus type 1 (HSV-1) grown in BSC-1 cells. Analysis of the DNA species synthesized in XC, R(B77) and normal rat (R-N) cells infected with HSV-1 revealed that the virus replicated in R(B77) and R-N cells, but failed to do so in XC cells. The virus particles adsorbed to the surface of the XC cells, but were unable to penetrate into the cytoplasm. Brief treatment with trypsin removed the virus particles adsorbed to the cell membrane. The HSV-1 particles synthesized in R(B77) cells were able to infect XC cells and to produce viral DNA and infectious virus progeny. These studies demonstrate that two steps are required for the infection of mammalian cells. The first step is the electrostatic adsorption of the virus particles to the cell membrane, and the second step is the interaction of the outer virus envelope with the host cell membrane. The latter step is specific and is dependent on the viral envelope.