Chloroplast DNA (cpDNA), containing 10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I(2) (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for 5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence.