It is generally accepted that the increase in choroidal thickness in response to myopic defocus in chicks acts to move the retina towards the image plane. It may also constitute part of the signal cascade in the visual regulation of eye growth. To test this, we used the nitric oxide synthase inhibitor l-NAME to inhibit the defocus induced choroidal thickening under two different visual conditions, and looked at the effects on ocular growth rate. Exp. 1: Deprivation/Vision: chicks were monocularly deprived of form vision with translucent diffusers from day 6 to day 9. In the middle of each day the diffusers were removed for 2 h. One group received an intravitreal injection of 30 μl l-NAME (16 μmole; n = 12) prior to the vision, a second group received injections of physiological saline (n = 11). Exp. 2: Recovery/Vision: chicks were made myopic by form deprivation from day 6 to day 10. On days 11 to 14 the diffusers were removed for 2 h per day for 4 days to allow eyes to “recover” from the myopia. One group received an injection of l-NAME prior to vision (n = 8), the other saline (n = 6). Refractive errors were measured with a refractometer at the start (days 6 and 11) and end (days 10 and 15, respectively) of both experiments. Ocular dimensions were measured with high frequency A-scan ultrasonography at the start and end, and on the third experimental day immediately before and after the period of vision. Choroidal retinoic acid synthesis was measured by HPLC. Finally, NO production and scleral proteoglycan synthesis were measured in eyes wearing positive lenses 6 and 24 h after an injection of l-NAME. l-NAME prevented the transient vision-induced choroidal thickening in both experiments. Furthermore, l-NAME inhibited the protective effect of brief daily vision: eyes became significantly more myopic than saline controls (exp. 1: −9 D vs −2.7 D; exp. 2: −0.9 D vs +4.3 D; p < 0.005 for both) and grew faster (change in lens-sclera: exp. 1: 295 vs 158 μm; exp. 2: 147 vs 39 μm; p < 0.05 for both). Notably, l-NAME inhibited the growth of the anterior chamber (exp. 1: 88 vs 185 μm; exp. 2: 147 vs 254 μm; p < 0.01 for both). Injections of l-NAME after the periods of vision had no effect on growth at the back of the eye, but still had an inhibitory effect on the anterior chamber. Retinoic acid levels in the drug-injected choroids were significantly decreased compared to controls. In eyes responding to positive lenses, l-NAME inhibited NO synthesis and disinhibited scleral glycosaminoglycan synthesis 6 h after the injection. In summary, preventing the transient vision-induced increases in choroidal thickness altered ocular growth rate in a consistent manner under two different visual conditions, in both preventing the vision-induced reduction in growth rate. This supports the hypothesis that visually-induced changes in choroidal thickness play a role in the visual regulation of ocular growth.