We have recently cloned the human Na+/myo-inositol cotransporter (SLC5A3) gene, mapped it to the q22 region on chromosome 21 and demonstrated significant expression in brain tissue. In certain cells, the SLC5A3 gene may be responsible for maintenance of a millimolar concentration gradient for myo-inositol (MI) as well as adaptation to changes in extracellular osmolality via transport-mediated alterations in levels of MI which functions as an alternate organic osmolyte. Depending on SLC5A3 expression, cells from patients with trisomy 21 may manifest increased MI levels, MI and Na+ flux and phosphoinositide levels or turnover. We determined the levels of MI in normal and trisomic 21 cultured human skin fibroblasts with and without hypertonic stress. After cells were grown in MEM(10 μM MI) with dialyzed fetal bovine serum ± 72 hour exposure to additional 25 mM NaCl, extracts were prepared and analyzed by 1H-NMR spectroscopy. The mean MI level in trisomy 21 cells was 144 ± 31 nmoles/mg protein (mean ± SEM, n=4) and in normals 62 ± 10(n=4). With hypertonic stress, the mean MI content was increased to 125± 29 and 214 ± 36 in normal and Down syndrome cells, respectively. Basal MI levels are increased in fibroblasts from patients with trisomy 21 (p < 0.05) but the MI adaptive response to hypertonic stress in trisomy 21 cells was comparable to controls (Δ = 63 vs 70). The physiological consequence of increased MI flux in the trisomy 21 cell bearing 3 copies of the SLC5A3 gene deserves further study.