The parasitic nematode Strongyloides ratti reproduces by both parthenogenesis and sexual reproduction, but its genetics are poorly understood. Cytological evidence suggests that sex determination is an XX/ XO system. To investigate this genetically, we isolated a number of sex-linked DNA markers. One of these markers, Sr-mvP1, was shown to be single copy and present at a higher dose in free-living females than in free-living males. The inheritance of two alleles of Sr-mvP1 by RFLP analysis was consistent with XX female and XO male genotypes. Analysis of the results of sexual reproduction demonstrated that all progeny inherit the single paternal X chromosome and one of the two maternal X chromosomes. Therefore, all stages of the S. ratti life cycle, X with the exception of the free-living males, are XX and genetically female. These findings are considered in relation to previous analyses of S. ratti and to other known sex determination systems.