The recently developed female leukophore-free (FLFII) strain of Japanese medaka (Oryzias latipes) carries DNA markers for the identification of genotypic sex. Information regarding genotypic sex is useful for tests in which endocrine-disrupting compounds may masculinize or feminize fish. In the present study, methods were developed to automate DNA extraction and profiling for rapid determination of genotypic sex. Adequate amounts of DNA were isolated by robotic extraction procedures from the caudal fin. New primers were developed to include an 18-base pair segment that is in the X chromosome of female medaka but is absent in the Y chromosome of male medaka. Automated profiling methods with 96-well plates permitted analysis of the genomic sex of medaka at rates of up to 500 fish/d. We investigated the sensitivity of the FLFII strain to the feminizing effects of the potent estrogen 17beta-estradiol (E2), and we compared this sensitivity to that of a wild strain that has been used widely in the study of endocrine-disrupting compounds. All FLFII medaka exposed to 1 microg/L of E2 (n = 50) had the female gonadal phenotype (i.e., ovaries), and all but one wild-strain medaka exposed to 1 microg/L of E2 (i.e., 47 of 48 fish) had the female gonadal phenotype, indicating that the FLFII and wild strains have approximately equal sensitivities to the feminizing effects of E2. Analysis of the genotype of FLFII medaka confirmed that 100% of fish with the male genotype had been feminized to the female gonadal phenotype. The FLFII strain is an excellent teleost model for detecting feminization or masculinization of fish, and automated methods can be used for rapid analysis of the genotypic sex of FLFII medaka.