We reported that hyperhomocysteinemia (HHcy) evoked by folate-depletion increases arterial permeability and stiffness in rats, and low-folate without HHcy increases arterial permeability in mice. In this study we hypothesized that HHcy independently increases arterial permeability and stiffness in mice. C57BL/6J mice that received rodent chow and water (Con, n=12) or water supplemented with 0.5% L-methionine (HHcy, n=12) for 18±3 weeks had plasma homocysteine concentrations of 8±1 and 41±1 µM, respectively (p<0.05), and similar liver folate (~12±2 µg folate / g liver). Carotid arterial permeability, assessed as dextran accumulation (ng/min/cm²) using quantitative fluorescence microscopy, was greater in HHcy (3.95±0.4) vs. Con (2.87±0.41) mice (p<0.05). Stress vs. strain curves generated using an elastigraph indicated that: 1) maximal stress (N/mm²); 2) physiological stiffness (low strain Young's Modulus, mN/mm); and 3) maximal stiffness (high strain Young's Modulus, N/mm) were higher (p<0.05) in aortae from HHcy vs. Con mice. Thus, chronic HHcy increases arterial permeability and stiffness. Carotid arterial permeability also was assessed in age-matched C57BL/6J mice before and after incubation with: 1) xanthine (0.4 mg/ml) / xanthine oxidase (0.2 mg/ml; X/XO) to generate superoxide anion (O₂^-); or 50 µM DL-homocysteine in the presence of: 2) vehicle; 3) 300 µM diethylamine NONOate (DEANO; a nitric oxide donor); or 4) 10^-3M 4,5-dihydroxy-1,3-benzene disulfonic acid [Tiron; a non-enzymatic intracellular O₂^- scavenger]. Compared to pre-incubation values, X/XO and DL-homocysteine increased (p<0.05) permeability by 66±11% and 123±8%, respectively. DL-homocysteine-induced increases in dextran accumulation were blunted (p<0.05) by simultaneous incubation with DEANO or Tiron. Thus, acute HHcy increases arterial permeability by generating O₂^- to an extent whereby nitric oxide bioavailability is reduced.