OriLyt, thecis-acting element of Epstein-Barr virus lytic origin of replication, consists of upstream and downstream components. The upstream component plays a dual role in transcription and replication. The downstream component contains a homopurine-homopyrimidine sequence which forms an H palindrome. We show that the downstream component can adopt a triple helix structure in vitro, that the 5' border of the homopyrimidine sequence is sensitive to P1 nuclease when carried by a supercoiled plasmid and that an oligonucleotide complementary to the homopyrimidine strand is taken up by a plasmid carrying the OriLyt H palindrome. We also show that all mutations which alter the H palindrome impair both oligonucleotide uptake and OriLyt-dependent replication. Interestingly, compensatory mutations which restore an H palindrome also restore oligonucleotide uptake by the mutated plasmids and their OriLyt-dependent replication. Thus, there is a strong correlation between the inability of the OriLyt H palindrome to form a non-B-DNA structure in vitro and impairment of OriLyt-dependent replication. This suggests that the presence of a non-B-DNA structure in the OriLyt downstream component is required for OriLyt-dependent replication.