SAPK3 (stress-activated protein kinase-3, also known as p38γ) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of α1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38γ and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38γ binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr²⁸⁷ and Ser²⁹⁰in vitro, and Ser²⁹⁰ in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38γ to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β but not SAPK3/p38γ, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38γ with SAP90. ERK2 also phosphorylates SAP90 at Thr²⁸⁷ and Ser²⁹⁰in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38γ co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38γ, a first physiological substrate described for SAPK3/p38γ and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein–protein interactions at the synapse in response to adverse stress- or mitogenrelated stimuli.