A fragment corresponding to most of the variable domain of the rabbit heavy chain (VH) was obtained by tryptic digestion of the midly reduced and aminoethylated heavy chain from rabbit antibody 3T72. The domain size peptide was purified by gel filtration and shown to extend between residues 11(Leu) and 122(Lys) of the heavy chain by sequence analysis. The molecular size of the fragment (approximately 11 000) was determined by gel filtration under denaturing conditions. Under nondenaturing conditions (20 mM sodium acetate, pH 5.5, 0.1 M NaCl), however, the fragment exists as a mixture of monomeric and dimeric species. The varable-domain fragment retains the allotypic determinants of the heavy chain (a1), as shown by double diffusion on agar plates and radioimmunoassay. Upon recombination of the heavy-chain variable-domain fragment with its homologous light chain, partial recovery of specific binding activity toward the SIII polysaccharide antigen was demonstrated. The method reported here is reproducible (with yields varying between 40 and 60%) and may provide a general method for obtaining the variable region of the heavy chain for antigen binding and allotypic and amino acid sequence studies.