To clarify the role of alkaline phosphatase (ALP) and ATPase in skeletal mineralization, we studied the effect of the anthelmintic drug, l-tetramisole (levamisole), a stereospecific inhibitor of ALP activity, and its inactive isomer, d-tetramisole (dexamisole), on in vitro calcification of rachitic rat cartilage. ALP activity in homogenized rachitic rat proximal tibiae was inhibited by l-tetramisole in a dose-dependent manner. Histochemical and electron microscopic cytochemical analysis of intact epiphyseal plate rachitic rat cartilage showed that 5 x 10(-2) M and greater concentrations of l-tetramisole (1) virtually abolished ALP activity, (2) moderately reduced ATPase activity, and (3) prevented in vitro cartilage calcification, while preserving the structural integrity of the matrix vesicles. Concentrations of d-tetramisole as high as 1 x 10(-1) M failed to inhibit ALP activity in tibial homogenates by more than 10 per cent and did not alter histochemical staining of enzyme activity or calcification in intact cartilage slices. Heating the cartilage slices destroyed ALP activity, prevented calcification, and disrupted matrix vesicle integrity. These data show that there is a close association between ALP activity and in vitro calcification of rachitic rat cartilage. In the absence of ALP activity, intact matrix vesicles do not promote calcification. Our data also suggest that some ATPase activity of rachitic rat cartilage may be distinct from ALP activity.