17-beta-estradiol (E2) is broadly believed to be the major circulating form of estrogen in almost all vertebrates. Its role in the stimulation of vitellogenesis in females is well established, and the assumption is that E2 stimulates other physiological and behavioural processes related, in particular, to reproduction. During incubation studies into steroid biosynthetic pathway preference in blue-tongued lizards, Tiliqua nigrolutea, we noted that while E2 was not produced, an alternative, highly polar steroid was synthesised in significant quantities. We have investigated this steroid further, using in vitro incubation with various steroid precursors, with products identified by a combination of TLC, HPLC with radiometric detection, UV spectrum comparisons and preliminary GC-MS. We know the steroid is more polar than E2, but less polar than estriol (E3). It is synthesised from pregnenolone (P5), probably via testosterone (T), but is not synthesised from androstenedione (AD). Gonadal production of this polar steroid varies with sex and reproductive condition: greater production is seen in both sexes during the spring reproductive period (greatest in vitellogenic females). We believe that this polar steroid is an alternative to E2 and could have important roles in reproductive physiology in this species.