Prostaglandin E(2) (PGE(2)) mediates the masculinization of adult sex behavior in rats in response to the surge in serum testosterone at approximately birth. Measures of behavioral masculinization correlate with a twofold increase in spinophilin protein and the density of dendritic spines in the medial preoptic area (POA). Of the four receptors for PGE(2), EP(2) and EP(4) are required for the masculinization of behavior by PGE(2). EP(2) and EP(4) couple to G(s)-proteins, activating protein kinase A (PKA). By using H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl) and Ht31, disruptors of PKA signaling, we have determined that PKA signaling is required for the masculinization of behavior by PGE(2). Glutamatergic signaling often mediates PGE(2) signaling; therefore, we tested whether inhibition of AMPA/kainate and metabotropic glutamate receptor (mGluR) signaling prevents PGE(2)-induced behavioral masculinization and whether activation of glutamate receptors mimics PGE(2). Females treated neonatally with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) plus LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] combined (AMPA/kainate and mGluR inhibitors, respectively) before PGE(2) did not exhibit as many mounts or intromission-like behaviors or initiate these behaviors as quickly as animals treated with PGE(2) alone. Animals neonatally treated with kainate, (+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) (type I mGluR agonist), or the two combined mounted as frequently and initiated mounting behavior as quickly as those given PGE(2). Ht31 does not prevent the masculinization of behavior by ACPD plus kainate cotreatment; rather, the coadministration of NBQX plus LY341495 prevents the forskolin-induced formation of POA dendritic spine-like processes. We conclude that PKA, AMPA/kainate, and metabotropic glutamate receptor signaling are necessary for the effects of PGE(2), that each receptor individually suffices to organize behavior, and that PKA is upstream of the glutamate receptors.