Procedures for separating and recombining the light-chain variable (VL) and heavy-chain variable (VH) domains of antibody Fv fragments have been applied to produce a recombinant Fv of the anti-digoxin antibody 26-10 that is 15N-labeled exclusively in the VL domain. Comparison of a two-dimensional 1H-15N heteronuclear single-quantum correlation (HSQC) spectrum of the reconstituted Fv with a HSQC spectrum of a fully 15N-labeled Fv sample reveals that all 1H-15N correlations of the VL domain align precisely in both spectra. Assignments for 105 of the 106 backbone HN groups of the VL domain within the reconstituted Fv have been obtained by analysis of three-dimensional nuclear Overhauser effect spectroscopy-HSCQ and total correlation spectroscopy-HSQC spectra with reference to assignments previously reported for the isolated VL domain. Chemical shift differences between the isolated VL domain and the VL domain within the Fv are moderately correlated with proximity to the surfaces of the VH domain and bound hapten (ouabain) as defined by X-ray crystallography and molecular modeling. These results demonstrate that nuclear magnetic resonance studies of reconstituted antibody Fv fragments, in conjunction with investigations of isolated antibody domains, can yield extensive resonance assignments for the Fv. This will facilitate detailed studies of antigen-antibody and domain-domain interactions.