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Testes from mature rats, rabbits, hamsters, guinea-pigs and dogs were perfused in vitro with added gonadotrophins to study the qualitative and quantitative aspects of oestrogen secretion by these species. After separation by high performance liquid chromatography (HPLC), the major oestrogen secreted by the testes of all five species was oestradiol-17β; lesser amounts of oestrone were also detected for all species. Secretion rates of oestradiol and testosterone were determined for one testis from each of six animals of each species after purification by HPLC. Oestradiol and testosterone secretion varied (P < 0·05) among species on both per testis and per gram of testis bases. The rabbit, which was a high secretor of oestradiol, was used in subsequent experiments to study the factors which affect short-term oestradiol secretion by perfused testes. Luteinizing hormone (NIAMDD-LH-S21) at 100 ng/ml significantly stimulated oestradiol secretion by 2·9-fold and increased testosterone secretion by 155-fold. Follicle-stimulating hormone (NIH-FSH-S11) had no significant effect on secretion of either steroid. In a time-course study in which LH stimulation was started after 45 min of control perfusion, the rates of increase in oestradiol and testosterone secretion over time were similar. Increasing amounts of testosterone added directly to the arterial perfusion medium (0–10 µg/ml) resulted in an increase in oestradiol secretion in the absence of gonadotrophins. Oestradiol secretion increased in a dose-dependent manner up to 1·0 µg testosterone/ml; there was no further stimulation of oestradiol secretion at 10 µg testosterone/ml. When testes were perfused with saturating concentrations of testosterone (10 µg/ml) neither LH nor FSH at 100 ng/ml increased oestradiol secretion above control values. It appears that the major factor affecting oestradiol secretion by in-vitro perfused rabbit testes is the amount of substrate (testosterone) available for aromatization.