The fragile X syndrome is one of more than a dozen genetic diseases attributed to the amplification of a trinucleotide repeat. Despite the number of these disease loci, relatively little is known about the mechanism(s) that cause a stable allele to become unstable. Population and family studies of the fragile X CGG repeat have identified a number of factors that may play a role in repeat instability including the number of AGG interruptions, purity of the 3' and 5' end of the repeat and cis-acting factors as related to haplotype background. However, studies that assess whether these factors have an impact on the rate and magnitude of change of the repeat are lacking, mainly due to the lack of an appropriate model system. Therefore, in order to dissect the factors involved in the initial mutations of the CGG repeat, small pool (SP)-PCR was performed on DNA derived from sperm and blood from seven unaffected males whose repeat sizes range from 20 to 33. Using the SP-PCR-derived data, regression analyses suggested that components of the repeat structure such as the number of interruptions and purity of the 3' end of the repeat are important determinants of germline repeat instability. In contrast, elements other than repeat structure, such as haplotype background, seemed to have an impact on somatic repeat instability. The factors identified for either cell type, however, explained only a small portion of the variance, suggesting that other factors may be involved in this process.