The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-Δ⁷-dafachronic acid isomer as the most potent compound, but the (25S)-Δ⁴-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-Δ⁷-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC₅₀ values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Δ⁴-dafachronic acids, with EC₅₀ values of 23 and 66 nm, respectively. The (25S)- and (25R)-Δ⁵-isomers were much less potent, with EC₅₀ values approaching 1000 nm, and saturated 5α- and 5β-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-Δ⁷-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Δ⁷-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25.