To have more insight into the mechanism of neuronal injury in phenylketonuria patients, gene expression profiles were studied in cell culture of embryonic rat cortical neurons induced by phenylalanine. Randomly chosen cortical cultures for 3 d were treated by 0.9 mmol/L phenylalanine for 12 h. Control group of the same batch was treated with the same volume of medium. Total RNA was extracted and hybridized with the Affymetrix gene chip U34 according to the protocol provided by the Affymetrix Company. Real-time PCR was used to further confirm the result. We found that the hybridization signals of 167 genes were increased among the total 1323 probes plotted on the chip. The 167 increased genes could be functionally categorized into signal transduction, neuron related, cytoskeleton, metabolism, ion channels, transcription factors, cytokines, and apoptosis related. Signals of 7 probes were decreased, which accounted to 0.5% of the total number. A series of genes that were not reported before were up-regulated by phenylalanine, including Ca(2+)/calmodulin-dependent protein kinase, Brain type II (CaMK II), Ras, P38 MAP kinase, L-voltage dependent calcium channel, some genes related to vesicle formation and transmitter release, some glutamate receptor subunits and glutamate transporters. According to the gene expression profile, it is likely that multi-processes are involved in the neuronal injury induced by high phenylalanine, such as the activation of the NMDR-Ca(2+)- CaMK II - Ras- P38 axis, the abnormality in neurotransmitter release. Our study also suggests that the excitatory neurotransmitter glutamate may play a role in the neural pathology of phenylketonuria.