The solution structure of the isolated V L domain of the anti-digoxin antibody 26-10 has been determined using data derived from heteronuclear multi-dimensional nuclear magnetic resonance (n.m.r.) experiments. Analytical ultracentrifugation and n.m.r. data demonstrate that the V L domain is only weakly associating ( K d = 2·5(±0·7) mM) and that it experiences a rapid monomer/dimer equilibrium under the n.m.r. experimental conditions. Therefore, the results reported here represent the first structure determination of an antibody V L domain in the absence of fixed quaternary interactions. The structure determination is based on 930 proton-proton distance constraints, 113 dihedral angle constraints, and 46 hydrogen bond constraints. Eighty initial structures were calculated with the variable target function program DIANA; of these 31 were accepted on the basis of satisfaction of constraints (no distance constraint violations >0·5 Å; target function < 3·0 Å 2). Accepted DIANA structures were refined by restrained energy minimization using the X-PLOR program. The 15 best energy-minimized DIANA structures were chosen as a representative ensemble of solution conformations. The average root-mean-square difference (r.m.s.d.) between the individual structures of this ensemble and the mean coordinates is 0·85(±0·10) Å for all backbone atoms and 1·29 (±0·10) Å for all heavy atoms. For β-strands A, B, C, D, E and F, the average backbone atom r.m.s.d. to the mean structure is 0·46(±0·06) Å. A higher-resolution ensemble, with all backbone atom and all heavy atom r.m.s.d.s to the mean coordinates of 0·54(±0·08) Å and 0·98(±0·12) Å, respectively, was obtained by X-PLOR simulated annealing refinement of the 15 energy-minimized DIANA structures. A detailed analysis of the original ensemble of 15 energy-minimized DIANA structures is presented, as this ensemble retains a broader, and possibly more realistic, sampling of conformation space. The backbone atom and all heavy atom r.m.s.d.s. between the mean energy-minimized DIANA structure and the X-ray derived coordinates of the V L domain within the Fab/digoxin complex are 1·05 Å and 1·56 Å, respectively. Subtle differences between the solution and X-ray structures occur primarily in CDR2, CDR3, β-strands A, F, and G, and localized regions of hydrophobic packing. Overall, these results demonstrate that the 26-10 V L domain conformation is determined primarily by intra-domain interactions, and that quaternary V L-V H association induces relatively minor conformational adjustments.