We investigated the production of oxytocin (OT) and oxytocin-neurophysin (bNpI) by bovine granulosa cells cultured in presence of 10% foetal calf serum, a condition known to induce spontaneous luteinization of these cells. The production of immunoreactive OT was significantly higher in the cultures of granulosa cells harvested from large follicles than in those derived from small follicles. Chromatography on Sephadex G-25 showed similar elution sites of ovarian and synthetic OT, while high performance liquid chromatography revealed two peaks of OT-immunoreactivity, one of which (+/- 65% of the total immunoreactivity) coincided with synthetic OT. In another experiment, we could observe a gradual increase of OT, bNp I and progesterone production by granulosa cells derived from large follicles, in relation with the incubation time. The mean molar ratio OT: bNp I was 2.2 +/- 0.5 (SEM), and we found a significant positive correlation between the production of OT and bNp I (r = 0.77; P less than 0.01) and between the production of OT and progesterone (r = 0.80; P less than 0.01). Furthermore, the cellular OT and bNp I content of large follicles-derived granulosa cells before culture was 4-5 times lower than the total amount of OT and bNp I produced during a 72-h incubation, suggesting an active synthesis of these peptides. These data show that bovine granulosa cells are able to produce OT and bNp I, probably by an active biosynthesis as observed in the hypothalamo-neurohypophysial system and that the granulosa productions of OT, bNp I and progesterone are closely related.