DNA sequence analysis and 5'-end mapping of mRNA were used to identify and clone DNA fragments which contain the presumptive promoter (Pgl) and poly(A) site (An) of the bovine herpesvirus 1 (BHV1) gl glycoprotein. To confirm the presence of these regulatory regions in the above fragments, they were cloned together with a chloramphenicol acetyltransferase (CAT) reporter gene into pUC19. The recombinant plasmid formed, pEC3, was capable of inducing CAT activity when transfected into bovine cells demonstrating that the Pgl-CAT-An sequence constituted a functional CAT expression cassette. The cassette was excised from pEC3, transferred to a plasmid insertion vector (pIV3A) and subsequently inserted into the thymidine kinase (tk) gene of BHV1. Insertion in either orientation, relative to the tk gene, gave rise to BHV1 recombinants which expressed CAT activity in infected cells. Analysis of RNA from infected cells indicated that CAT transcripts were present in multiple species of RNA. This unexpected result was found to reflect temporal shifts in promoter and poly(A) site usage during infection. Although the poly(A) site which forms part of the expression cassette was used extensively early in infection, most CAT transcripts synthesized at late times read through this promoter-proximal site and terminated at the distal site normally used for tk mRNA synthesis.