GnRH neurosecretion is subject to regulation by insulin, IGF-I, leptin, and other neuroendocrine modulators whose effects may be conveyed by activation of phosphoinositide 3-kinase (PI3K)-mediated pathways. It is not known, however, whether any of these regulatory actions are exerted directly, via activation of PI3K in GnRH neurons, or whether they are primarily conveyed via effects on afferent circuitries governing GnRH neurosecretion. To investigate the role of PI3K signaling in GnRH neurons, we used conditional gene targeting to ablate expression of the major PI3K regulatory subunit, p85alpha, in GnRH neurons. Combined in situ hybridization and immunohistochemistry confirmed reduction of p85alpha mRNA expression in GnRH neurons of GnRH-p85alpha knockout (KO) animals. Females of both genotypes exhibited estrous cyclicity and had comparable serum LH, estradiol-17beta, and FSH levels. In male GnRH-p85alphaKO mice, serum LH, testosterone, and sperm counts were significantly reduced compared with wild type. To investigate the role of the other major regulatory subunit, p85beta, on the direct control of GnRH neuronal function, we generated mice with a GnRH-neuron-specific p85alpha deletion on a global betaKO background. No additional reproductive effects in male or female mice were found, suggesting that p85beta does not substitute p85 activity toward PI3K function in GnRH neurons. Our results suggest that p85alpha, and thus PI3K activity, participates in the control of GnRH neuronal activity in male mice. The sex-specific phenotype in these mice raises the possibility that PI3K activation during early development may establish sex differences in GnRH neuronal function.