Recently, zebrafish has quickly risen as a model species for functional analysis of the brain-pituitary-gonad axis. However, one of the hurdles for such work in this popular model organism is the small size of its pituitary gland, which makes it difficult to investigate the regulation of pituitary hormone expression and secretion in vitro. To provide a solution to this problem and demonstrate the value of zebrafish in reproductive endocrinology, the present study was undertaken to establish a primary pituitary cell culture followed by investigating the regulation of FSHbeta (fshb), LHbeta (lhb), and GH (gh) expression by a variety of neuroendocrine, endocrine, and paracrine factors. All the factors examined influenced the expression of fshb, lhb, and ghin vitro except epidermal growth factor (EGF) despite the expression of its receptor egfr in the pituitary. Acting in a similar manner, gonadal steroids (estradiol and testosterone) stimulated both fshb and lhb, but had no effect on gh. In contrast, all other factors tested (gonadotropin-releasing hormone, GnRH; pituitary adenylate cyclase-activating polypeptide, PACAP; activin/follistatin, and insulin-like growth factor I, IGF-I) exhibited distinct effects on the expression of the three target genes studied, suggesting roles for these factors in the differential regulation of two gonadotropins and growth hormone and therefore the gonadotrophic and somatotrophic axes.