Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ER alpha), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t) ER alpha, tAR, and tPR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tER alpha, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tER alpha protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tER alpha, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones.