ATP-dependent ⁴⁵Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO₄ and oxalate. In the absence of divalent anions, there was a transient ⁴⁵Ca accumulation, lasting only a few minutes. Addition of PO₄ did not change the initial accumulation but added a second stage that increased with PO₄ concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 µM), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 250 µM), and DIDS (3-5 µM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 µM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO₄ cotransport. An oxalate-supported ⁴⁵Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO₄ and oxalate had activating and inhibitory effects that could be explained by PO₄ inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO₄ in brain endoplasmic reticulum.