Osteopenia is a frequent and early complication of Rett syndrome. This study aimed to evaluate the usefulness of Quantitative Ultrasonography (QUS) at phalanxes in the assessment and monitoring of bone status in Rett patients. We studied 109 girls (10.1+/-6.1 years; range 3-25 years) and 101 age-matched controls. Serum calcium (Ca), bone alkaline phosphatase (B-ALP), parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD) and QUS parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT) were measured. At baseline both QUS parameters and 25OHD levels were significantly lower in Rett patients than in controls. Serum 25OHD was inversely correlated with serum PTH and BTT Z-score and BTT Z-score was significantly lower (p<0.05) in the girls with a 25OHD serum levels<or=9 ng/ml. Moreover, multiple regression analysis showed that BTT was significantly influenced by 25OHD levels. The design of the study was to carry out a twelve monthly evaluation of QUS and biochemical parameters for 3-years. 82 Rett patients completed the 36 month longitudinal study. During the study period in the patients, who at baseline were non ambulatory, BTT and AD-SoS Z-score presented the tendency to markedly decrease; in contrast, mild changes of the QUS parameters were observed in patients with severe or mild-moderate ambulatory impairment. Significant difference of BTT Z-score and AD-SoS Z-score among the 3 groups of patients categorized on the basis of ambulatory performance were observed at years 2 and 3. The AD-SoS Z-score at the end of the study period were significantly influenced by the changes in ambulatory performance and the use of anticonvulsant therapy. In conclusion our results suggest the usefulness of QUS parameters at phalanxes in the monitoring of bone status in Rett patients. Low levels of 25OHD and ambulatory impairment play a key role in the skeletal deterioration in Rett girls.