The bioactive lipid platelet‐activating factor (PAF) accumulates in brain during injury, seizures and ischemia and may, in addition, be significant in AIDS dementia and in other neurodegenerative diseases. We have used plasma‐type recombinant PAF acetylhydrolase (rPAF‐AH) to test the hypothesis that PAF accumulation is involved in early events leading to neuronal apoptosis during excitotoxic neuronal injury. Neuronal cultures were labeled with FITC‐12‐dUTP (TUNEL technique) and propidium iodide, digitized using fluorescence microscopy and a chilled 3CCD color camera, and analyzed with 2D graphics analysis software. N‐methyl‐D‐aspartate (NMDA) (50 μM, 2 hr) induced a 2.5‐fold increase in apoptosis of hippocampal neurons compared with controls when analyzed 24 hr after NMDA treatment. Hippocampal neurons receiving rPAF‐AH (20 μg/ml) before, during, and after NMDA treatment demonstrated a concentration‐dependent neuroprotective effect which resulted in 47% and 30% neuroprotection against 50 and 100 μM NMDA, respectively. The noncompetitive NMDA receptor antagonist MK‐801(300 nM) completely inhibited apoptosis caused by NMDA. The neuroprotective effect of rPAF‐AH against NMDA‐induced apoptosis was confirmed using as additional criteria, histone release, electron microscopy, and DNA laddering. Neuroprotection elicited by rPAF‐AH demonstrates that PAF is an injury mediator in NMDA‐induced neuronal apoptosis and that the recombinant protein is potentially useful as a therapeutic approach. J. Neurosci. Res. 53:677–684, 1998. © 1998 Wiley‐Liss, Inc.