Intestinal motility disorders are more common in women of childbearing age who are prone to iron deficiency anemia. The neurotransmitters nitric oxide (NO) and acetylcholine (ACh) play a key role in ileal smooth muscle relaxation and contraction, respectively. Iron-containing heme is known to be a cofactor for nitric oxide synthase (NOS), the enzyme responsible for NO production. Therefore we tested the hypothesis that iron deficiency would downregulate ileal NOS activity without affecting the ileum’s response to ACh. Twelve adult female prairie dogs were fed either an ironsupplemented (Fe+) (200 ppm) (n = 6) or an iron-deficient (Fe-) (8 ppm) (n = 6) diet for 8 weeks. Ileal circular muscle strips were harvested to measure responses to ACh and electrical field stimulation. Under nonadrenergic noncholinergic (NANC) conditions, Nω-nitro-L-arginine (L-NNA), an NOS inhibitor, and VIP10-28, a vasoactive intestinal peptide (VIP) inhibitor, were added prior to electrical field stimulation. NANC inhibitory responses are expressed as a percentage of optimal relaxation from EDTA. The excitatory response to ACh was similar in both groups (1.1 ± 0.3 N/cm2 vs. 1.5 ± 0.3 N/cm2, P = 0.45). The inhibitory response to electrical field stimulation under NANC conditions was greater in the Fe+ group (34.7 ±2.9%) compared to the Fe-group (23.9 ±3.2%; P <0.01). L-NNA eliminated the inhibitory response in the Fe+ group (0.02 ± 0.02%) but not in the Fe-group (8.38 2 2.15%; P <O.Ol). VIP10-28 led to greater relaxation in the Fe+ animals (45.8 ± 6.6%) than in the Fe-animals (23.4 ±5.8%; P <0.05). Both L-NNA and VIP10-28 had no inhibitory response (0.02 ± 0.02%) in the Fe+ animals, whereas the Fe-animals had some residual inhibition (2.54 ±1.04%; P <0.05). These data suggest that ileal NANC relaxation is due to NOS and that iron deficiency results in (1) decreased NANC relaxation, (2) a compensatory relaxation due to a non-NOS, non-VIP mechanism, and (3) a normal excitatory response. We conclude that iron deficiency suppresses ileal NOS activity.