We constructed Fab libraries of bacteriophage-displayed HCDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of HTrp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from HCDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward HCDR3, thereby providing room for C16 substituents in the region of HCDR1.