This paper reports on the optimal incubation conditions for in vitro transcription in isolated yeast mitochondria. The mitochondrial RNA polymerase has an absolute requirement for all four nucleoside triphosphates and Mg2+. The enzyme is inhibited by Mn2+, Ca2+, Cd2+ and KCl while low concentrations of NaCl stimulate activity. Transcription is maximal in Tris/HCl buffer at pH 7.9 and the optimum incubation temperature is 28 degrees C.