An experiment was conducted on 48 ewes during follicular and luteal phases of the oestrous cycle to determine the effect of a 5-day lupin grain supplementation (500 g/day) on folliculogenesis, plasma concentrations of glucose, insulin, FSH and oestradiol-17β (E2), follicular fluid concentrations of glucose, E2, androstenedione and progesterone and the levels of P450 aromatase and insulin receptor substrate 1 (IRS-1), -2 and -4 in theca and granulosa cells. Average weight did not differ between lupin-fed and control groups. The numbers of follicles were increased (P<0.05; χ(2)) in the lupin-fed group. The plasma concentrations of glucose (P<0.05; ANOVA) and insulin (P<0.001; ANOVA) were higher in lupin-fed ewes. The plasma concentrations of FSH were not different but those of E2 were decreased (P<0.001) in the lupin-fed group. Both the follicular fluid concentration of E2 (P<0.05) and the level of P450 aromatase in granulosa cells (P<0.05; ANOVA) were decreased in the lupin-fed group, but only during the follicular phase. The level of P450 aromatase in granulosa cells was positively correlated with the concentration of E2 in follicular fluid (r=0.820; P<0.001; ANOVA). The levels of IRS-1 and -2 in theca and granulosa cell lysates were increased in the lupin-fed group. These data suggest that insulin has a local role in the control of folliculogenesis and is likely to be a mediator of the effects of dietary energy intake on ovulation rate. We suggest that insulin acting through IRS proteins mediates the reproductive actions of insulin in the follicle and that IRS-1 and -2 are nutritionally regulated mediators of the action of insulin in the follicle.