Calbindin D(28K) binds 3 mol of terbium per mol of protein. To determine which of six EF-hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF- hand 2 (RCaBP Δ2), the other lacking EF-hands 2 and 6 (RCaBP Δ2,6), and the third containing only EF-hands 3 and 4 (RCaBP Δ1,2,5,6), and examined their binding properties by fluorescence techniques. Full-length calbindin D(28K) and RCaBP Δ2 and RCaBP Δ2,6 bound 3 mol of terbium per tool of protein with high affinity. Thus, EF-hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C. Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites. To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a Δ1,2,5,6 RCaBP construct. The truncated protein RCaBP Δ1,2,5,6 contained a single terbium- binding site. Analysis of the terbium binding to RCaBP Δ1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D(28K) are responsible for terbium binding.