Recent evidence has suggested a role for phosphorylation in the regulation of ligand-gated ion channels. We have recently shown (Ruiz-Gómez, A., Vaello, M., Valdivieso, F., and Mayor, F., Jr. (1991) J. Biol. Chem. 266, 559-566) that the inhibitory glycine receptor (GlyR) alpha subunit is phosphorylated in vitro by protein kinase C (PKC). In this report we further show that alpha subunits of the GlyR can also be phosphorylated by cAMP-dependent protein kinase (PKA) in an in vitro assay. Moreover, incubation of intact rat spinal cord neurons with specific PKC or PKA activators leads to increased phosphorylation of the GlyR alpha subunits, strongly suggesting a physiological role in its functional modulation. The role of protein phosphorylation in modulating GlyR channels was explored in Xenopus oocytes injected with poly (A)+ mRNA isolated from nervous tissue. The treatment of oocytes with phorbol esters or dibutyryl cAMP resulted in a decrease or an enhancement, respectively, of glycine-evoked currents. Our results show that the GlyR can be phosphorylated in vivo in response to activation of either PKC or PKA with opposite functional consequences, suggesting that neurotransmitters affecting the activity of such kinases could profoundly alter glycine-mediated neuronal signaling and modulate synaptic efficacy.