Wild-type rat androgen receptor and four of its deletion mutants were produced in insect cells using the baculovirus expression system. Inclusion of androgen, but not estrogen, progesterone, or glucocorticoid, in culture medium increased the yield of soluble androgen receptors, although the majority of receptors still remained in the insoluble form (Xie, Y.-B., Sui, Y.-P., Shan, L.-X., Palvimo, J. J., Phillips, D. M., and Jänne, O. A. (1992) J. Biol. Chem. 267, 4939-4948). The wild-type receptor interacted with an androgen response element (ARE) with a 2-6-fold higher affinity (KD = 0.5 nM) than mutants with deletions outside the DNA-binding domain (delta 40-147, delta 38-296, delta 46-408, and delta 788-902 mutants), suggesting that sequences flanking the DNA-binding region influence the stability of receptor-DNA complexes. Changes in spacing (n = 3) between the two ARE half-sites by a single nucleotide (n - 1, n + 1) or by 10 bases (n + 10) abolished the full-length receptor's ability to form stable complexes with DNA. Binding to AREs with altered spacing could not be restored by antisera against the N-terminal domain of the receptor that stabilize androgen receptor-DNA interactions with many naturally occurring strong and weak AREs. Methylation interference and 1,10-phenanthroline copper footprinting analyses revealed that the receptor binds to DNA as a dimer. Dimer formation was demonstrated directly by mixing full-length and delta 46-408 mutant receptors, which resulted in the formation of heterodimeric receptor-DNA complexes. The half-time of dissociation of the wild-type receptor from a consensus ARE sequence was about 3 min at 22 degrees C. Collectively, androgen receptor binds to DNA with properties similar to, but not identical with, those of glucocorticoid receptor, indicating that regions outside the DNA-binding domain are important to ensure androgen specificity of transcriptional regulation.