Herpes simplex virus type 1 (HSV-1) is a common human herpesvirus that has a sero-prevalence between 70-80% in the adult population and can lead to oral/genital lesions and viral encephalitis. Infected cell protein (ICP) 27 (ICP27) is an essential, 512-residue HSV-1 protein that performs many different functions during infection including but not limited to the regulation of viral gene expression, participating in viral mRNA export and modulating the cellular interferon response pathway. The majority of previous studies of this viral protein have been conducted in Vero cells, a line of African green monkey kidney cells. Here we have examined the phenotype of viral ICP27 mutants in other cell lines and primary cultures. We identified one mutant, d1-2, which shows a striking cell type-dependent growth defect in that it can partially replicate in Vero cells and some other cells but cannot replicate at all in many human cells including primary fibroblasts. Analysis of d1-2 infections showed that its restricted replication is associated with markedly decreased expression of the viral ICP8 gene and aberrant formation of viral replication compartments. These data suggest that viral DNA replication is tightly blocked in restrictive cells. Using a plasmid transfection/virus complementation strategy, we demonstrate that the cell-type dependent replication phenotype of HSV-1 mutants maps to the N-terminus of ICP27, specifically to residues 12-20. Together, our data indicate that ICP27 function is dependent on host cell type and that HSV-1 studies in Vero cells may not fully model natural human infections.