Sex determination results from the translation of an initial switch into a complex phenotype that integrates morphology, physiology and behavior to define an organism as male or female. Despite the fundamental importance of this pathway, a wide range of both environmental and genetic sex determination mechanisms is found in nature. Closely related species may have different sex chromosome complements and use completely different mechanisms to determine sex, suggesting that these developmental pathways can evolve very rapidly. How can the rapid evolution of sex determination mechanisms and sex chromosomes occur? Stickleback fish provide an exciting system to address these questions. Recently diverged stickleback species have different sex chromosome complements and may also use different sex-determination mechanisms. Powerful genetic and genomic tools, which make it possible to clone and identify the genes underlying sex determination pathways, have been developed for sticklebacks. This goals of this proposal are to: (1) characterize the sex-determination region found on an evolving sex chromosome in threespine sticklebacks (Gasterosteus aculeatus) through genetic, cytogenetic and physical mapping of the region; (2) molecularly identify the master sex-determination gene in threespine sticklebacks through both loss-of-function and gain-of-function approaches; and (3) compare the chromosomal locations of the master sex-determination gene in closely related species of sticklebacks to determine if sex-determination hierarchies and sex chromosomes have evolved independently in these species. These studies are initial steps towards a long-term goal of comparing sex determination pathways in closely related species to explore the evolution of this fundamental developmental pathway and to determine its effects on the morphology, physiology, and behavior of the organism.