A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using SalI, PstI, KpnI and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with 32P-dCTP nick-translated KpnI probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chloroplast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants.