The effect of beta-aminopropionitrile (BAPN) on lipid synthesis and 14CO2 production from 14C-acetate by rat calvariae in vitro was investigated by biochemical, histochemical, and autoradiographic techniques. Exposure of bones to 5 mM BAPN throughout a 5-hour incubation period resulted in little effect on lipid synthesis when compared with control bones. Higher BAPN concentrations, up to 100 mM, during this short incubation period resulted in enhanced lipid radioactivity for most of the identified lipid classes. Longer preincubation periods of 20 hours and low BAPN (20 mM) concentrations resulted in enhanced lipogenesis. As BAPN concentrations above 20 mM were employed, lipogenesis decreased to such a level that some lipid classes failed to become labeled with 14C-acetate. 14CO2 production from 14C-acetate by a rat calvariae was affected by BAPN concentration and incubation time in a pattern similar to that of lipid synthesis. Histochemistry demonstrated neutral fat in the osteogenic cells and phospholipids in osteoblasts and the osteoid matrix. Autoradiographic localization of 14C-acetate revealed that the radioactivity was in the osteogenic cells and that this activity was markedly increased after pretreatment of the calvariae with BAPN. This study shows that some concentrations of BAPN enhance lipogenesis by rat calvariae and supports the earlier suggestion that lipids play a role in biologic calcification.