To determine the relationship between alkaline phosphatase (AP), 1,25(OD)2D3 and bone formation in vivo, we have examined the effects of levamisole, a stereospecific inhibitor of AP on bone formation and on 1,25(OH)2D3-stimulated bone mineralization in the mouse. Normal mice were injected daily with levamisole at doses of 40 and 80 mg/kg/b.w. The compound was given alone or in combination with 1,25(OH)2D3 infusion (0.05 micrograms/kg/d) for 7 days. Treatment with levamisole alone inhibited the serum AP activity (mainly of skeletal origin in mice) by 18.4 and 61.3% for the low and high dose respectively. No deleterious effect on body growth, tibia length, and bone cells population was detected. The moderate inhibition of AP activity produced by the lower dose of levamisole alone (18.4%) or in combination with 1,25(OH)2D3 (37.9%) was associated with a reduced endosteal matrix apposition rate (MaAR) determined by double 3H-proline labeling method. This effect was related to a levamisole-induced fall in serum phosphate. Despite the moderate inhibition of AP activity, the mineral apposition rate (MiAR) determined by the double tetracycline labeling method remained normal. Moreover, 1,25(OH)2D3 infusion still resulted in increased MiAR which was stimulated to the same extent as in the absence of levamisole. By contrast, the more severe inhibition of AP activity induced by 80 mg/kg of levamisole alone (61.3%) or in combination with 1,25(OH)2D3 (45.8%) inhibited both the MaAR and the MiAR and prevented the stimulatory effect of 1,25(OH)2D3 on bone mineralization. The data show that AP activity affects the bone matrix and mineral apposition rates in vivo and that severe inhibition of AP activity inhibits the 1,25(OH)2D3-induced stimulation of bone mineralization in the mouse.