Glutamate-mediated excitotoxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The astroglial glutamate transporter EAAT2 plays a major role in maintaining low levels of extracellular glutamate in the central nervous system. Multiple EAAT2 mRNA transcripts have been described, but those retaining intron 7 or skipping exon 9 are reported to be specific to the motor cortex, spinal cord, and cerebrospinal fluid of ALS patients. We sought to verify these findings using a TaqMan (Elmer Biosystems, Warrington, UK) real-time reverse transcriptase polymerase chain reaction assay, which provides a sensitive and reliable quantitative measure of EAAT2 transcript copy ratios. We analyzed RNA extracted from frozen postmortem tissue from affected and unaffected central nervous system regions dissected from 17 sporadic ALS patients, 7 Alzheimer's disease patients, and 19 control subjects. We have demonstrated unequivocally that intron 7 retaining and exon 9 skipping variants can be detected in all individuals and in all central nervous system regions studied. The mean ratio of "variant" to "normal" transcripts did not differ significantly between patient and control groups. Although our assay could detect transcript concentrations in cerebrospinal fluid as low as 10 pg/ml, none were detected in 17 ALS and 8 control samples. We conclude that ALS is not associated with elevated levels of EAAT2 transcripts retaining intron 7 and skipping exon 9. An alternative explanation must be sought for the disturbance of glutamate homeostasis reported in ALS.