In order to study DNA replication and expression in wheat mitochondria our laboratory has been seeking to develop a system that supports DNA synthesis and transcription, either in isolated mitochondria from wheat embryos or in a mitochondrial lysate from the same source deprived of endogenous DNA in vitro. We have characterized some of the enzymes involved in the DNA synthesis and transcription process. In this study we describe a DNA topoisomerase activity.Broken mitochondria from wheat embryos can actively relax negatively supercoiled DNA (pBR322, pAT153, etc...). The enzyme is intramitochondrial: the activity is detected only when intact organelles are broken by non-ionic detergent. Most of the topoisomerase activity found in the broken mitochondria is recovered in the mitochondrial lysate. It is stimulated by Mg(2+) and has an optimum salt concentration, KCl or NaCl, between 50 mM and 100 mM. ATP has no effect on this activity. Ethidium bromide, berenil, novobiocine and nalidixic acid, compounds currently used to characterize DNA topoisomerases, do not effect the relaxation of supercoiled DNA by the wheat mitochondrial activity. On the other hand N-ethylmaleimide has a strong inhibitory effect indicating that sulfhydryl groups are essential for enzyme activity. The molecular weight of the enzyme as determined by glycerol gradient sedimentation, is about 110 kd. Another important feature of the mitochondrial lysate DNA topoisomerase is the ability to relax positively supercoiled DNA, a property of eukaryotic topoisomerases I.