Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid-one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERalpha, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.