Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, approximately 1.5x10(10)) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V(H)) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, approximately 2.5x10(10)), highly diversified phage-displayed human V(H) library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V(H)s were with different CDR2 origins (six of seven groups of V(H) germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V(H)s against viral and cancer-related antigens; all selected V(H)s were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.