Previous studies have shown that exogenous steroid hormones can affect sex determination in reptiles with temperature-dependent sex determination (TSD). These studies have also suggested that the sensitivity of TSD to exogenous steroids may vary with incubation temperature. The majority of these studies, however, have utilized incubation temperatures producing all males or all females in the control groups, rather than temperatures which produced mixed sex ratios in control groups. The goals of the current study were to examine the effects of steroids on sex determination in a turtle (Trachemys scripta) at temperatures which produced mixed sex ratios in the control groups. Collectively, the results of single-treatment experiments indicate that at incubation temperatures producing mixed sex ratios in control groups, (1) estradiol-17β, tamoxifen, norethindrone, and testosterone all showed a similar "type" of effect (i.e., feminizing) as in previous studies utilizing male-producing temperatures, (2) sex determination has significantly increased sensitivity to estradiol-17β in comparison to its effect at temperatures producing all males, and (3) sex determination is sensitive to the masculinizing effects of dibydrotestosterone (DHT) (in previous studies utilizing female producing temperature DHT did not affect sex determination). Last, a set of double-treatment experiments was performed in which eggs received both estradiol-17β and DHT treatments. No significant increases in the production of males were detected. Significant increases in the production of females were detected, but only in the groups receiving the highest dosage of estradiol-17β (1.0 μg). This contrasts the results of the single-treatment experiments in which lower dosages of estradiol-17β were effective (0.1 and 0.01 μg), thus suggesting that DHT in some way decreases the effectiveness of estradiol-17β. Further, a number of hatchlings in the double-treatment experiments developed intersex gonads (i.e., the gonads had well-developed medullary and cortical regions), suggesting that cortical and medullary development of the gonads are not mutually exclusive. © 1995 by Academic Press, Inc.