The androgen receptor (AR) is a ligand-activated transcription factor required for male sex development and virilization and contributes to prostate cancer initiation and progression. High affinity androgen binding triggers conformational changes required for AR transactivation. Here we characterized naturally occurring AR gene mutations in the region of activation function 2 (AF2) that decrease or increase AR transcriptional activity by altering the region bounded by AF2 and the ligand binding pocket without affecting equilibrium androgen binding affinity. In the androgen insensitivity syndrome, germ line AR mutations increase the androgen dissociation rate and reduce AR FXXLF motif binding and the recruitment of steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs. In prostate cancer, somatic AR mutations in AF2 or near the bound ligand slow androgen dissociation and increase AR stabilization and coactivator recruitment. Crystal structures of the AR ligand binding domain bound to R1881 and FXXLF or LXXLL motif peptide indicate the mutations are proximal to the AF2 bound peptide, adjacent to the ligand pocket, or in a putative ligand gateway. The results suggest a bidirectional structural relay between bound ligand and coactivator that establishes AR functional potency in vivo.