Sulfated polyanions can be used to rapidly induce and maintain single-cell suspensions of BTI-TN5B1-4 insect cells, a cell line which clumps in suspension. Elimination of cell clumping results in a significant increase involumetric yield of the baculovirus expression vector system. Sulfated polyanions, however, inhibited baculovirus infection of BTI-TN5B1-4. Data from binding studies and fusion assays suggest that the inhibition of infection was not due to the observed reduction in viral attachment rate but to inhibition of viral membrane fusion in the endosome. The three most effective polyanions for inducing single cells are dextran sulfate, pentosan sulfate, and polyvinyl sulfate. At concentrations required for single-cell formation, dextran sulfate and pentosan sulfate did not affect viral infection at multiplicities of infection greater than one plaque forming unit per cell. In contrast, polyvinyl sulfate blocked viral infection even at a high multiplicity of infection of 20 plaque-forming units per cell. To bypass this inhibition, polyvinyl sulfate can be removed by resuspending the cells in fresh medium before virus addition, and then added back to the cell suspension after a substantial amount of virus has been internalized. Alternatively, polyvinyl sulfate can be neutralized with a polycation before virus addition, and an equivalent amount of polyvinyl sulfate added back after most of the virus has been internalized. We present a simple mathematical model of the attachment and entry of baculovirus in BTI-TN5B1-4, which can be used to design appropriate infection regimens.