Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 α, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of γ-1 genes, (ii) induction of expression of γ-2 genes, (iii) down regulation of expression of α and β genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of γ-1 proteins but appeared to be unable to efficiently express γ-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates γ-1 gene expression and a second one required for γ-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral β genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.