Effective repression of cI transcription from P_RM by the bacteriophage λ CI repressor requires binding sites (O_L) located 2.4 kb from the promoter. A CI tetramer bound to O_L1.O_L2 interacts with a tetramer bound near P_RM (O_R1.O_R2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant O_L3 operator and the weak O_R3 operator overlapping P_RM are juxtaposed so that a CI dimer at O_L3 can cooperate with a CI dimer binding to O_R3. Here we show that O_L3 is necessary for effective repression of P_RM and that the repressor at O_L3 appears to interact specifically with the repressor at O_R3. The O_L3-CI-O_R3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the λ system as a powerful tool for examining long-range gene regulatory interactions in vivo.