One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5α-dihydrotestosterone. Further analysis of the AR Leu701 variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel of structurally related steroids to study which of the OH groups at positions 11β, 17α, and 21, which discriminate cortisol from progesterone, underlie the differential responses to both hormones. The results showed that the 17α-OH group was essential for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated a conserved H-bonding network involving the steroidal 17α-OH group, His701 or Gln701, and the backbone of Ser778. This network is absent in Leu701 and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17α-OH group. Our results indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser778, and the steroidal 17α-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone and cortisol of AR mutants, including the prostate cancer mutant L701H.