Fragile X syndrome, the most common form of mental impairment, is caused by expansion of a (CGG)n trinucleotide repeat element located in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Repeat expansion is known to influence both transcription and translation; however, the mechanisms by which the CGG element exerts its effects are not known. In the current work, we have utilized 5'-RLM-RACE to examine the influence of CGG repeat number on the utilization of transcription start sites in normal (n<55) and premutation (54<n<200) cell lines of both non-neural (lymphoblastoid) and neural (primary astrocyte) origin. Our results demonstrate that, in both neural and non-neural cells, transcription of the FMR1 gene is initiated from several transcription start sites within a approximately 50 nt region that lies approximately 130 nt upstream of the CGG repeat element. For normal alleles, most transcripts initiate from the downstream-most start site, close to the single position identified previously. Surprisingly, as the size of the CGG repeat expands into the premutation range, initiation shifts to the upstream sites, suggesting that the CGG element may act as a downstream enhancer/modulator of transcription. The shift in start site selection for both neural and non-neural cells indicates that the effect is general. Furthermore, the correspondence between start site utilization and the degree of elevation of FMR1 mRNA suggests that a substantial fraction of the increased message in the premutation range may derive from the upstream start sites.