The study of intracellular Ca2+ regulation usually requires using calcium chelators to adjust [Ca2+]. We examined the effects of these chelators on calcium accumulation in microsomes and saponin-permeabilized synaptosomes to assess their influence on apparent transport properties. At a fixed free Ca2+ of 0.6 microM, increasing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) and total Ca2+ enhanced ATP-dependent 45Ca sequestration in synaptosomes and microsomes. The EGTA-Ca complex did not change the maximal initial calcium uptake rate or maximal steady-state accumulation. Rather, EGTA/Ca increased the apparent affinity of the microsomal transporter for Ca2+. The presence of the organic anion transport inhibitor probenicid (2.5 mM) had no effect on 45Ca accumulation in the presence of EGTA. Replacing part of the Ca2+ with Ni2+ but maintaining [Ca2+] approximately constant reduced 45Ca uptake, suggesting that the Ni-EGTA complex did not stimulate 45Ca transport. Our results imply that EGTA is not actively transported across the endoplasmic reticulum membrane, nor does the divalent ion-bound form of EGTA change the properties of the transporter. EGTA, and other mobile calcium chelators with similar structures, e.g., 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, indo 1, and fluo 3, may increase calcium uptake by delivering more Ca2+ to its transport site.