Intracellular factors that regulate nitric oxide (NO) synthesis represent important targets in tumor progression. Overexpression of dimethylarginine dimethylaminohydrolase (DDAH), which metabolizes the endogenous inhibitors of NO synthesis asymmetric dimethylarginine and N-monomethyl-L-arginine, results in C6 gliomas with enhanced growth rate compared with wild type. To investigate the effects of DDAH on tumor vascular morphogenesis in vivo, we have measured the transverse relaxation rates R(2)* and R(2) in clone D27 gliomas overexpressing DDAH and C6 wild-type gliomas using intrinsic susceptibility magnetic resonance imaging (MRI), sensitive to changes in endogenous [deoxyhemoglobin], and susceptibility contrast-enhanced MRI using the intravascular blood pool contrast agent NC100150, and we compared the results with fluorescence microscopy of the tumor uptake of the perfusion marker Hoechst 33342. The baseline R(2)* was significantly faster in the D27 tumors, consistent with a greater vascular development (P < 0.02, ANOVA). There was no significant difference between the response of the two tumor types to hypercapnia (5% CO(2)/95% air), used as a probe for vascular maturation, or hyperoxia (5% CO(2)/95% O(2)), used as a probe for vascular function. NC100150 increased the R(2)* and R(2) rates of both tumor types and demonstrated a significantly larger blood volume in the D27 tumors (P < 0.02, ANOVA). This correlated with a significantly greater uptake of Hoechst 33342 in the D27 tumors compared with C6 wild-type tumors (P < 0.02, ANOVA). Despite the increased tumor blood volume, the Delta R(2)*/Delta R(2) ratio, an index of microvessel size, showed that the capillaries in the two tumor types were of a similar caliber. The data highlight the potential of susceptibility MRI-derived quantitative end points to noninvasively assess tumor angiogenesis, and in this regard, the use of intravascular blood pool contrast agents such as NC100150 appears very promising. Overexpression of DDAH results in increased neovascularization of C6 gliomas in vivo. The lack of significant difference in hypercapnic/hyperoxic response between the C6 and D27 tumors and the similar vessel caliber are also consistent with a role for DDAH in the initial stages of vasculogenesis.