Histologic assessment of the gonads to detect intersex has become a valuable end point in reproductive toxicologic testing for fish, and many studies have solidly linked intersex with exposure to endocrine active substances (EAS). An assumption in such studies is that spontaneous intersex does not occur in control fish. Using historical data derived from toxicologic tests with Japanese medaka (Oryzias latipes), we report a retrospective study in which we identified 54 individual instances of intersex (testicular oocytes or ovarian testicular tissue) in control medaka from 15 of 41 selected toxicologic studies. These studies, comprised of previously unpublished data, had been conducted at three geographically distant laboratories, each of which utilized unique water sources, employed somewhat different culture protocols, and maintained distinct medaka breeding colonies. During our histologic examinations, we also identified three germ cell neoplasms that had been inadvertently diagnosed as intersex. In the present report, we review potential causes of intersex, discuss possible reasons why spontaneous intersex has rarely been reported, and propose suggestions for the judicious interpretation of intersex results in medaka studies involving EAS.