The role of multiple gonadotropin-releasing hormone receptor (GnRH-R) types in the regulation of gonadotropic and nongonadotropic cells remains speculative. To address this issue, we developed a technology integrating laser-captured microdissection of single digoxigenin-labeled pituitary cells coupled with real-time quantitative PCR to examine the expression profiles of three endogenous GnRH-R types (R1, R2, and R3) in immature and mature males of tilapia Oreochromis niloticus. Here, in addition to gonadotropes (luteinizing and folicle-stimulating hormone, FSH), we show GnRH-Rs are also present in lactotropes, somatotropes, thyrotropes, melanotropes (melanocyte-stimulating hormone, MSH), corticotropes and somatolactin cells. Subpopulations of pituitary cells express single (42.9%), multiple (32.4%) or lack (24.7%) GnRH-Rs. In immature males, the percentage of FSH cells containing combinations of GnRH-Rs was significantly higher (R1؉R2: 24%, P < 0.05; R1؉R2؉R3: 25%, P < 0.01) than in mature males, whereas the percentage showing only R1 and R1 and R3 transcripts (P < 0.05) was higher in mature males. Significantly greater copies of R1 and R3 transcripts were found in MSH cells of immature and mature males, respectively (P < 0.05). GnRH-R transcripts in other pituitary cells (lactotropes, R1 and R2; somatolactin cells͞thyrotropes͞corticotropes, R1, R2, and R3) were significantly higher in mature males (P < 0.05) but were unaltered in somatotropes and luteinizing hormone cells. Thus, FSH and MSH cells are required for both reproductive states, whereas other pituitary cells are recruited only during testicular maturation. The differential expression of GnRH-Rs in gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity of mechanisms controlling normal sexual development.G protein ͉ in situ hybridization ͉ tilapia G onadotropin-releasing hormone (GnRH) is now recognized as a family of 16 multifunctional neuropeptides in vertebrates (1-3). It is well documented that all vertebrate species ranging from fish to humans possess two (hypothalamus, GnRH1; midbrain, GnRH2) or, as in recently derived teleosts, three GnRH types (caudal olfactory bulbs, GnRH3) (1-3). GnRH1, GnRH2, and GnRH3, in addition to stimulating gonadotropes (follicle-stimulating hormone, FSH; luteinizing hormone, LH), are potent regulators of somatotropes [growth hormone (GH) cells], lactotropes [prolactin (PRL) cells], and somatolactin (SL)-containing cells in teleosts (4-9). Because GnRH exerts its actions through binding to GnRH receptors (GnRH-Rs) (10), it is, therefore, conceivable that the three GnRH types have their respective cognate receptors expressed in different pituitary cells. Multiple GnRH-Rs have been cloned (10) and their transcripts identified in LH and GH cells (11), and GnRH-R proteins in LH, GH, and PRL cells (4) of teleosts. However, the distribution of GnRH-R transcripts in other endocrine cell types of the adenohypophysis has never been evaluated. Besides, it is unknown whether transcripts of multip...