By means of enzymatic and autoradiographic techniques, it has been demonstrated that, 24 hr after a single dose of the antitumor amino acid N-phosphonacetyl-L-aspartic acid (PALA), (400 mg/kg i.p.; 1.15 mmol/kg) to C57BL x DBA/2 F1 mice, the agent accumulates in bone to a concentration of approximately 400 microM; this is 3000 times greater than the Ki of PALA for its target enzyme, aspartate carbamoyltransferase. However, disproportionately low inhibition of enzyme activity was demonstrated in homogenates of bone from these recipients, suggesting that the drug was sequestered from its target in this tissue. Autoradiography of sections of femoral shafts from mice treated with 14C-labeled drug demonstrated that autoradiogram density due to [14C]PALA equivalents was confined to the bony matrix, with no label above background resolvable in bone marrow. Following in vivo administration of PALA (400 mg/kg i.p.), the half-life of the drug in the bone was approximately 23 days. In vitro, with equilibrium dialysis at pH 7.4, it was demonstrated that: (a) normal pulverized and decalcified bone bound PALA with capacities of 3.5 nmol/mg and 0.1 nmol/mg bone, respectively, at a PALA concentration of 5 mM; (b) binding of PALA to normal bone reached saturation at a concentration of 200 mM; and (c) PALA functions as a solubilizer of bone at concentrations above this. Since administration of PALA was shown to produce long-lasting inhibition of aspartate carbamoyltransferase in liver and tumor and since its ultimate half-life in the plasma of mice, following a single 400-mg/kg administration of the drug, is 8 days, it is suggested that bone serves as a reservoir from which PALA is released at a slow rate into plasma and other tissues.