To characterise the pattern of the transcriptional regulation of equine herpesvirus type 1 (EHV-1)UL24by regulatory proteins, we identified two distinct promoter regions and two transcription initiation (Tci) sites located upstream of the UL24 open reading frame (ORF). The ORF proximal promoter exhibited highercis-activity than that of the distal one. Contrary to the former, the latter performed its function dependent on an initiator (INR) due to its lack of a TATA box. Our results showed that the EHV-1 regulatory proteins EICP0, EICP22 and ETIFtrans-activated the two promoters, whereas IEP and IR2P displayed negative regulation. In summary, the regulatory proteins exhibited similar regulatory patterns for the two distinct promoters of EHV-1UL24.