The immediate-early (IE) gene of pseudorabies virus (PRV) has recently been sequenced for two virus strains. To investigate IE gene regulation and to examine the genome segment reported to encode latency-related transcripts in opposite polarity to the IE gene, sequence analysis has been extended by 5 kb from each end of the IE gene. The IE promoter (P1) was found to be more complex than previously recognized: it consisted of nine imperfect repeats, each containing five to six different consensus elements for transcription factor binding. A second promoter (P2) was discovered downstream of the IE gene. It contained numerous octamer consensus sequences (ATGCAAAT) and recognition sites for transcription factor Sp1; specific binding of nuclear proteins to four Sp1 sites was detected. An open reading frame (ORF3) bordering on P2 was identified, oriented antiparallel to the IE gene. Potential enhancer elements (E3 and E4) were isolated by the enhancer trap technique. Linked to P1 and a CAT indicator gene, E3 acted as an enhancer and E4 as a silencer. The PRV IE gene product repressed transcription from its own promoter and activated the SV40 early promoter. The transactivating virion protein Vmw65 of HSV1 had an opposite effect on these promoters.