A transcriptionally active chromosome has been isolated in highly purified form from choroplasts of Euglena gracilis, It contains chloroplast DNA, DNA-dependent RNA polymerase, and other proteins. Transcription occurs at low levels of endogenous DNA, and is indifferent to high levels of exogenous DNA. RNA chain elongation continues for several hours in vitro, and RNA chain initiation, determined by [gamma-32P]ATP incorporation, is continuous for at least 1 h in vitro. Maximal rates for RNA synthesis require only a divalent cation and the four ribonucleoside triphosphates. Apparent Km values for adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate are 4.0, 0.6, 2.5, and 2.3 muM, respectively. As would be expected for a DNA-dependent RNA polymerase, RNA synthesis is inhibited by actinomycin D. However, rifampicin and streptolydigin, inhibitors of procaryotic RNA synthesis, and alpha-amanitin, an inhibitor of eucaryotic nuclear RNA polymerases II and III, do not inhibt the RNA synthesis reaction. Heparin, which is a potent inhibitor of the initiation of RNA synthesis by a nontemplate bound RNA polymerase, also does not inhibit RNA synthesis. Isolation of transcriptionally active chromosomes should prove to be a useful method to study the mechanism of selective RNA transcription of eucaryotic chromosomes.