Rett syndrome, an X-linked neurodevelopmental disorder, is a major cause of mental retardation in females. Recent genetic analyses have revealed that mutations in the methyl-CpG-binding protein gene encoding MeCP2 are associated with Rett syndrome. In this study, we used transient expression systems to investigate the functional significance of mutations seen in patients with Rett syndrome. Missense mutations in the methyl-CpG-binding domain were analyzed by the transfection in mouse L929 cells and Drosophila SL2 cells. The L929 cells were utilized to investigate the effects of mutations on the affinity for heterochromatin, where methylated CpG dinucleotides are extremely enriched. The SL2 cells were utilized to analyze their effects on transcriptional repression activities. R106W and F155S mutations led to the substantial impairment of MeCP2 functions, showing the loss of accumulation of the mutated protein to mouse heterochromatin and the reduction of the transcriptional repressive activity in Drosophila SL2 cells. Intriguingly, the R133C mutant retained the functionality equivalent to MeCP2 in these analyses. On the other hand, the T158M mutation exhibited the intermediate level of the impairment of functions in both analyses. Thus, these functional assays are useful to evaluate the consequences of mutation in the methyl-CpG-binding domain of MeCP2 and provide an insight into the relationship between the genotype and the severity of Rett syndrome.