A new calcium-selective ionophore N,N-dioctyl-3alpha,12alpha-bis(N-heptyl-N-methylcarbamoyl-methoxyacetamidoacetoxy)-5beta-cholan-24-amide (denoted as BACA), was synthesized, and its potentiometric performance has been evaluated in comparison with that of the best known calcium-selective neutral carriers, ETH 129 and ETH 1001. The 1H NMR spectra of BACA titrated with Ca(SCN)2 suggest that BACA forms a 1:1 complex, tweezing a calcium ion between the two parallel diamide groups substituted on a rigid bile acid frame. The calcium-selective membrane based on BACA was less selective to calcium (log K(Ca2+ j)POT = -4.2, -4.2, -4.6, and -4.8, respectively, for j = Mg2+, Li+, Na+, and K+) than those based on ETH 129 (log K(Ca2+ j)POT = -4.4, -4.3, -5.4, and -5.4, respectively, in the same order) and ETH 1001 (log K(Ca2+ j)POT = -4.4, -4.4, -5.4, and -5.4), implying that BACA forms a weaker calcium complex than the other two ETH compounds. In our experimental conditions, potentiometrically determined complex formation constants of calcium-selective neutral carriers (log beta(Ca2+ L)) were 15.2, 14.0, and 8.6 for ETH 129, ETH 1001, and BACA, respectively. A slightly reduced calcium selectivity of BACA, however, affects the anionic interference-free calcium-selective membrane; the BACA-based membrane exhibits a Nernstian response up to 10(-1) M Ca2+ in the presence of lipophilic anions (e.g., SCN-, ClO4-, salicylate, and I-) and anionic surfactant, whereas the ETH 129- and ETH 1001-based ones suffer from serious anionic interference showing a curvature or leveled off response over about 10(-4) M. It was demonstrated that such a trade off does not affect the analytical performance of BACA-based electrode in most applications, including clinical analysis.