Yttrium and indium complexes of 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetylbenzylamine (DOTA-BA) and 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetyl-R-(+)-alpha-methylbenzylamine (DOTA-MBA) were prepared in order to study solution structures of (90)Y- and (111)In-labeled DOTA-biomolecule conjugates. (90)Y and (111)In complexes M(L) (M = (90)Y and (111)In; L = DOTA-BA and DOTA-MBA) were prepared from the reaction of MCl(3) with DOTA-BA and DOTA-MBA, respectively, in ammonium acetate buffer. A reverse phase HPLC method revealed that both (90)Y and (111)In complexes show only one radiometric peak in their radio-HPLC chromatograms. It was also found that (111)In(DOTA-BA) and (111)In(DOTA-MBA) are more hydrophilic than their corresponding (90)Y analogues, suggesting different coordination spheres in (111)In and (90)Y complexes of the same DOTA conjugate. Complexes M(L) (M = Y and In; L = DOTA-BA and DOTA-MBA) were prepared and characterized by HPLC, LC-MS, and NMR ((1)H and (13)C) methods. The HPLC concordance experiments for (90)Y(DOTA-MBA)/Y(DOTA-MBA) and (111)In(DOTA-MBA)/In(DOTA-MBA) show that the same complex is prepared at both tracer and macroscopic levels. The NMR data ((1)H and (13)C) clearly demonstrates that Y(DOTA-BA) and Y(DOTA-MBA) exist in solution as one predominant isomer. VT NMR data ((1)H and (13)C) show that In(DOTA-BA) and In(DOTA-MBA) are fluxional at room temperature while Y(DOTA-BA) and Y(DOTA-MBA) become fluxional only at elevated temperatures. The fluxionality of these complexes is due to rapid rotation of acetate/acetamide chelating arms and inversion of ethylenic groups of the macrocyclic ring.