Systematic investigation of the stability of mono-, bis-, and tris-terbium(III) complexes with dipicolinic acid is reported. The formation constants were extracted from emission titration, data. For this purpose, a mathematical formalism for two complexation models was developed. Comparative studies were conducted by variation of the acidity of the medium (from H-0 = -2 to pH = 7), the buffer composition (i.e., containing seven different anions), and the salt concentration (from 0.04 to 1 M). It became evident that elevating the pH and lowering the salt concentration were important, not only for increasing the values of the formation constants, but also for forming polynuclear Tb-DPA complexes. Inhibition of the latter phenomenon was observed when acetate or sulfate was present. The main purpose of the present investigation was to establish optimum conditions for conducting terbium(III)-mediated emission assays for bacterial endospore detection.