The following investigation was undertaken to study the location in the dental plaque and calculus of certain enzyme activities and to compare the patterns obtained with those of the normal hard tissue formation. Supragingival and subgingival calculus attached to the root surfaces of 30 extracted teeth was studied. The root with its deposits was frozen rapidly in a mixture of hexane and solid CO2 (-75 degrees C). From the frozen block, sections were cut and incubated for histochemical demonstration of lactate dehydrogenase, alkaline phosphatase and acid phosphatase. The plaque seemed to be stratified with regard to enzyme activity. Three different layers could be identified. In the basal layer, approximately 100 microns thick, enzyme activity was low. Lactate dehydrogenase activity could be identified in some sections, but no phosphatase activity. In the middle layer lactate dehydrogenase, alkaline phosphatase and acid phosphatase activities were found in most of the sections. The superficial layer usually showed lactate dehydrogenase but not always acid or alkaline phosphatase activities. The results of the present investigation may suggest that the mineralization of the dental plaque is not only a passive mineralization of dead bacteria, but also an active process promoted by enzymes in the covering bacterial layers.