Fifty-seven individuals of the European cyprinid fish species Scardinius erythrophthalmus were sexed by gonad histology, and their karyotypes were analyzed by Giemsa staining and C-banding. The chromosome number in somatic metaphase plates was 2n = 50. Karyotypes of all gonadal male animals were identical, consisting of 48 small meta- or submetacentric chromosomes and a pair of large metacentric chromosomes (ZZ males). Of the 33 gonadal females, 16 had a karyotype similar to the males' (ZW' females) and 17 had a heteromorphic pair of chromosomes, including a large metacentric Z chromosome and a small acrocentric W chromosome (ZW females). H-Y antigen typing with cells obtained from a variety of tissues revealed that the homogametic ZZ males were H-Y negative and the heterogametic ZW females were H-Y positive in all tissues tested. In contrast, ZW' females showed no expression of the H-Y antigen in somatic cells but were H-Y positive in ovarian cells. These results suggest that the ZW/ZW'/ZZ sex chromosomes of S. erythrophthalmus represent an unusual system of sex determination and that H-Y antigen expression is coupled with the heterogametic sex. Whether the ZW' individuals are indeed heterogametic females or actually sex-reversed ZZ males, in which chromosomal sex determination is overruled by external factors, is discussed.