Aromatase is a steroidogenic enzyme catalyzing the production of estrogens and is important for the proper development and function of the reproductive system. The lineage of cyp19a1 (ovarian-type aromatase)-expressing cells in the developing gonad was analyzed using a transgenic medaka (Oryzias latipes) that recapitulates endogenous cyp19a1 expression with EGFP fluorescence. Our results show that cyp19a1-expressing cells arise in the ventral stromal cells of the developing female gonad, then expand anteriorly as the gonadal region extends anteriorly. These cells become located close to the developing follicles, and are distinguishable from the P450c17-I-expressing theca cells. In the adult ovary, the expression of P450c17-I and cyp19a1 are mutually exclusive in the outer theca-cell layer. Cyp19a1 expression in the granulosa cells is found only in the population of large follicles. These observations demonstrate two types of theca cells in the medaka ovary. We also show that the maintenance of cyp19a1-expressing cells depends on germ cells.