Bovine herpesvirus 1 (BHV-1) encodes four immediate-early (IE) proteins. The transactivators BICP0 and BICP4 are key regulatory elements in viral replication, and circ is a myristylated virion component, whereas BICP22--originating from a spliced 1.7-kb transcript synthesized with dual IE and late kinetics--has not yet been characterized as a protein. In this study, Western blot and immunofluorescence analysis using antisera against a C-terminal oligopeptide revealed major 50-kDa and minor 35-kDa species of BICP22, predominantly located in the nuclei of BHV-1 infected cells. In transient expression assays, BICP22 acted as transrepressor protein on viral promoters of different kinetic classes, e.g. the IE promoter of the BICP4/BICP0 gene, early promoter of the BICP0 gene, and late promoter of the gC gene. The BICP22 gene promoter itself was not repressed by BICP22; it could be dissected into a proximal region stimulated by BICP0 and a distal region stimulated by BHV-1 alpha-transinducing factor. Replacement of the BICP22 promoter by cytomegalovirus IE promoter revealed an additional posttranscriptional level of regulation whereby more BICP22 accumulated in cells when functional BICP0 was present. Interplay of BICP22 and BICP0 might involve the recently described nuclear domains (ND10) and ubiquitin-dependent pathway.