This study was designed to investigate the locus of action of different gonadotrophins on the stimulation of multiple follicular development and superovulation in heifers. In addition follicular populations and steroiodogenic potential of the follicles (>10 mm diameter) at different stages of the superovulatory treatment were studied. Experiment 1 was carried out to investigate the effect of three gonadotrophins (pregnant mare serum gonadotrophin, PMSG; human menopausal gonadotrophin, hMG-Pergonal 75 i.u. FSH, 75 i.u. LH per ampoule; and urofollitrophin UF-Metrodin 75 i.u. FSH per ampoule) on multiple follicular development and hormonal profiles. Experiments 2 and 3 were designed to study follicular populations and steroiodogenic potential of the follicles in superovulated heifers and to compare these parameters with control heifers.