R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.