A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.