Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions. The lambda Int protein is required for both directions, but Xis for excision only. To investigate the substrate requirements for directional control, we have characterized two mutations of the phage attachment site that are defective in integrative but not excisive recombination. Both of these mutations produce the same base change in the P'3 binding site for Int protein 79 base-pairs from the center of the crossover region for site-specific recombination. We infer that differential utilization of this distant binding site is crucial for directional control of recombination.