This study assessed the relationships of urinary D-chiro-inositol and myo-inositol excretions to indices of whole-body glucose tolerance and total content and tyrosine phosphorylation of the insulin receptor (activation) in skeletal muscle of older nondiabetic subjects. Fifteen adults (age, 65 +/- 8 years; body mass index, 27.9 +/- 3.3 kg/m(2) [mean +/- SD]) completed duplicate assessments of oral (75-g oral glucose tolerance test [OGTT]) and intravenous (300 mg/kg body weight intravenous glucose tolerance test) glucose tolerance challenges and 24-hour urinary D-chiro-inositol and myo-inositol excretions. Skeletal muscle (vastus lateralis) biopsies were obtained at minute 60 of the OGTTs. Subjects with higher urinary D-chiro-inositol excretion had higher insulin (rho = 0.51, P < or = .05) and C-peptide (rho = 0.56, P < or = .05) area under the curves, and lower insulin sensitivity index (rho = -0.60, P < or = .05) during the intravenous glucose tolerance test. The urinary myo- to D-chiro-inositol ratio was also inversely related to insulin area under the curve (rho = -0.59, P < or = .05). Urinary D-chiro-inositol (rho = -0.60, P < or = .05) and myo-inositol (rho = -0.60, P < or = .05) were inversely related to tyrosine phosphorylation of the insulin receptor (phosphotyrosine 1162/1163), but not total content of the insulin receptor during the OGTT. The apparent relationships were modestly weakened when adjustments were made for sex. These findings support previous research linking higher urinary D-chiro-inositol excretion with a progressive decline in whole-body glucose tolerance. This is the first report to link higher urinary D-chiro-inositol excretion to a blunted activation of skeletal muscle insulin receptor signaling in older nondiabetic subjects.