1. The activities and relative 3-oxoacyl-CoA substrate specificities of oxoacyl-CoA thiolase were determined in a large number of animal tissues. The relative activities with different 3-oxoacyl-CoA substrates varied widely in different tissues and, in addition, the activity as measured with acetoacetyl-CoA (but not with other longer-carbon-chain acyl-CoA substrates) was activated by K(+). 2. These properties were due to the presence, in different proportions in each tissue, of three classes of thiolase, all of which use acetoacetyl-CoA as substrate but which have different intracellular locations and substrate specificities and which differ also in kinetic and chromatographic behaviour. 3. Cytoplasmic thiolase activity was found to be widely distributed among different tissues and was due to an acetoacetyl-CoA-specific thiolase. This cytoplasmic activity was found to account for a significant proportion of the total tissue activity towards acetoacetyl-CoA in several tissues, and especially in the brain of newborn rats. 4. Mitochondrial thiolase activity towards acetoacetyl-CoA was due to two different classes of enzyme whose relative amounts varied with the tissue type. An oxoacyl-CoA thiolase of general specificity for the acyl-CoA substrate constituted one class, the other being a specific acetoacetyl-CoA thiolase that differed from its cytoplasmic counterpart in being greatly stimulated by K(+). 5. This activation by K(+) made it possible to calculate the tissue contents of mitochondrial acetoacetyl-CoA thiolase and mitochondrial oxoacyl-CoA thiolase from measurements of activity with acetoacetyl-CoA in tissue extracts under defined conditions. 6. The properties and the different thiolases and their tissue distribution is discussed with respect to their possible roles in metabolism.