We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.