Abstract 1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2. The mtDNA from freshly removed thyroids was isolated by buoyant density centrifugation in ethidium bromide CsCl gradients after DNAase treatment of the mitochondrial pellet. Typically, more than 90% of the recovered mtDNA was found in the lower band, indicating a low rate of nicking during isolation. A sample of the total mtDNA (upper and lower bands) was examined by electron microscopy after preparation by the aqueous protein film technique. 3. The frequency of circular dimers generally ranged from 0.1 to 0.3%. However, in an mtDNA sample from cow thyroid, the frequency of circular dimers was 0.6% (0.9% if circular dimers occurring in catenanes are included), differing significantly from the frequency of these forms in bull thyroid, 0.1%. A small but significant variability also occurred in the frequency of catenanes ranging from 2 to 8% in the different groups; this variation is within the limits usually observed in normal tissues. 4. These observations indicate that thyroids, like other normal tissues examined so far, have a low content of circular dimers. A high frequency of these forms seems to be the trademark of some genetically and physiologically abnormal cells such as certain established cell lines, virus-transformed cells and malignant or otherwise pathological tissues.