We previously provided evidence for a model of herpes simplex virus type 1 (HSV-1) entry by a cascade of interactions between components of the virion envelope and cellular plasma membrane (Fuller and Lee, 1992, J. Virol. 66, 5002-5012). In this report we have determined entry kinetics of wild-type HSV-1 into two highly susceptible cell lines to further explore the contributions of viral or cellular factors to entry. Penetration rates of preattached virus varied among several common laboratory HSV-1 strains into one cell line. However, entry kinetics varied substantially for a single strain into highly susceptible HEp-2 or Vero cells under identical conditions. Plaquing efficiencies and sensitivity to heparin also significantly differed between these cells. Kinetics of entry that included virus attachment and penetration showed that the cell-specific effects can be explained by two distinct phases of attachment that occurred before penetration, but differed in duration on both susceptible cell lines. Initial attachment of virus is resistant to removal with phosphate-buffered saline, but sensitive to removal with buffer containing heparin. This is followed by a second type of attachment that is heparin resistant, but still sensitive to extracellular inactivation. We conclude that although undefined factors unique to individual wild-type HSV-1 laboratory strains affect entry kinetics, entry of any one strain is greatly influenced by interactions of virus with specific cell components during at least two distinct phases of attachment before penetration. Moreover, the second phase to stabilize binding seems to be the rate-limiting event in entry. Since major differences in the amounts or sulfation patterns of heparan sulfate were not detected, differences in the surfaces of HEp-2 and Vero cells that influence the kinetics and efficiency of HSV-1 entry are likely in the fine structure of heparan sulfate or in the presence and quantity of other unidentified receptors.