The objective of this study was to determine the minimum light intensity directed at a single eye required to suppress melatonin (MT) levels to concentrations observed under indoor lighting systems in dairy cows. Following a 14-d environmental conditioning period comprising 8 h of light, 16 h of dark (LD8:16), where mean (±SD) white light intensity levels by day (08:00 – 16:00) at eye level were 237 ± 68 lx, 5 nonlactating Holstein-Friesian cows were exposed to each of the following light intensities for 8 additional hours (16:00 – 00:00) using a 5 × 5 Latin Square design; <1 lx; 70 lx; 125 lx; 175 lx and 225 lx. Light was administered via head worn masks fitted with LEDs emitting short wavelength blue light (465 nm) to the right eye. Each treatment night was followed by a break night, where the animals again received LD8:16. Two days after completion of the different light intensity treatments, all cows were exposed to the indoor lighting system until 00:00 (LIGHTS ON). Blood samples were collected from indwelling jugular catheters at 16:00, 17:00, 18:00, 20:00, 23:00, 00:00 and 01:00 on treatment nights and at 18:00 and 22:00 on break nights. Plasma samples were assayed for MT by radioimmunoassay. MT data were log-transformed, and analyzed using mixed models with treatment, period, hour and treatment × hour as fixed effects and cow as a random effect. A dose-dependent effect of treatment on mean circulating MT concentrations (and 95% CI) between 16:00 and 00:00 was observed [9.1 (6.8, 12.2), 4.9 (3.7, 6.6), 4.4 (3.2, 5.8), 3.3 (2.5, 4.4), 1.8 (1.4, 2.5) and 1.9 (1.4, 2.5) ng/mL for 0, 70, 125, 175, 225 lx and LIGHTS ON treatments, respectively]. Only the 225 Lux mask treatment did not differ from LIGHTS ON, and hence 225 lx is the minimum light intensity required. Future studies will examine the effect of photoperiod manipulation in grazing animals.