The dorsomedial periaqueductal gray (dmPAG) is a mesencephalic area and has numerous functions including cardiovascular regulation. Because nitric oxide (NO) is present in the dmPAG, here we investigate, the probable cardiovascular effect of NO in the dmPAG. Five groups (n = 6 for each group) were used as follows: (1) control; (2) L-NAME (NG -nitro-L-arginine methyl ester, a NO synthase inhibitor, 90 nmol); (3) L-arginine (L-Arg, a precursor for NO, 60 nmol); (4) Sodium nitroprusside (SNP, a NO donor, 27 nmol); and (5) L-Arg + L-NAME. The cardiovascular parameters were recorded by a Power Lab device after cannulation of the femoral artery. Drugs were injected using a stereotaxic instrument. The changes (∆) in systolic blood pressure (SBP), mean arterial pressure (MAP), and heart rate (HR) were calculated at different times and compared to the control group. Microinjection of L-NAME significantly increased ∆SBP, ∆MAP, and ∆HR more than saline (from p < 0.05 to p < 0.001). L-Arg only significantly increased ∆HR (p < 0.05). In the L-Arg + L-NAME group, the above parameters also significantly increased (from p < 0.01 to p < 0.05) but not as significantly as with L-NAME alone. Microinjection of SNP significantly decreased ∆SBP and ∆MAP more than in the control and L-NAME groups (from p < 0.01 to p < 0.001), but ∆HR did not change significantly. The results indicated that NO in dmPAG has an inhibitory effect on cardiovascular responses in anesthetized rats.