Gamete self-incompatibility in the hermaphrodite tunicate Ciona intestinalis is a useful system with which to study self-nonself recognition. We have used in vitro fertilization of oocytes isolated from the gonad of Ciona intestinalis to identify the cellular source of self-sterility elements present on the egg envelopes. Here we show for the first time that self-discrimination, which occurs on the egg vitelline coat, is established there in late oogenesis and is contributed or controlled by products of the overlying follicle cells. The acquisition of self-sterility by the oocyte is prevented by the ionophore monensin, which suggests that the follicle cell self-sterility controlling factor is a glycoprotein.