We have detected an unusual polar steroid as a major end product of gonadal steroid biosynthesis in male and female blotched blue-tongued lizards, Tiliqua nigrolutea. In an investigation of the steroidogenic pathways in blue-tongued lizards, we incubated gonads of both male and female lizards with tritiated pregnenolone and identified the products of steroid biosynthesis using thin layer chromatography and high performance liquid chromatography with radiometric detection: gonads from both reproductively active and quiescent lizards were examined. The proportion of total radioactive substrate converted to the unknown polar steroid was greater in individuals with hypertrophied gonads (males (N=4) 28.1%; females (N=4) 45.3%) than in those with regressed gonads (males (N=4) 5.9%; females (N=4) 33.3%). Properties of the unknown steroid were identical across all incubations. Incubation time-course information, thin layer and high performance liquid chromatography properties of the polar steroid are presented. The steroid is more polar than 17β-estradiol, which was not detected, but less polar than all tri-hydroxylated estrogens to which it was compared. The possibility of an alternative estrogen to 17β-estradiol in some reptiles is discussed.