A co-transfection method was used to identify regions of the Marek's disease virus (MDV) genome that inhibit plaque formation when introduced along with total DNA from MDV-infected chicken embryo fibroblasts into secondary chicken embryo fibroblasts. Co-transfections involving MDV plasmids containing the BamHI-D or BamHI-H regions of the genome inhibited plaque formation more than 10-fold. The inhibitory region was localized to a 222-base-pair region that contains a sequence homologous to the consensus origin of replication of alphaherpesviruses. The region also contains several potential transcriptional regulatory elements, including two CCAAT boxes, one Sp1 binding site, and an octamer element. The sequence of this region has been reported previously. Transfection inhibition was also observed for the BamHI-I2 region, although the effect was weaker.