The short-wavelength (blue) light sensitivity of human non-visual responses is recognised as being melanopsin-based. However, whether melanopsin is the sole factor in determining the efficacy of a polychromatic light source in driving non-visual responses remains to be established. Monochromatic (λ max 437, 479 and 532 nm) and polychromatic (colour temperature: 4000 K and 17000 K) light stimuli were photon matched for their predicted ability to stimulate melanopsin, and their capacity to affect nocturnal melatonin levels, auditory reaction time and subjective alertness and mood was assessed.Young, healthy male participants aged 18 -35 years (23.6 ± 3.6 yrs; mean ± SD; n = 12) participated in 12 overnight sessions that included an individually timed 30 min light stimulus on the rising limb of the melatonin profile. At regular intervals before, during and after the light stimulus subjective mood and alertness were verbally assessed, blood samples were taken for analysis of plasma melatonin levels and an auditory reaction time task (psychomotor vigilance task: PVT) was performed. Proc GLM repeated measures ANOVA analysis revealed that significantly lower melatonin suppression was observed with the polychromatic light conditions (4000 K and 17000 K) compared to the 'melanopsin photonmatched' monochromatic light conditions (p < 0.05). In contrast, whilst subjective alertness significantly increased across the course of the light exposure it was significantly lower under the 479 nm monochromatic light condition compared to the 437 and 532 nm monochromatic and both polychromatic light conditions. The demonstration that the melatonin suppression response to polychromatic light was significantly lower than predicted by the melanopsin photosensitivity function suggests that this function is not the sole consideration when trying to predict the efficacy of broadband lighting. The different spectral sensitivity of the subjective alertness and melatonin suppression responses may imply a differential involvement of the cone photopigments. An analysis of the photon densities in specific wavelength bands for the polychromatic lights used in this and our previous study suggests that the spectral composition of a polychromatic light source, and particularly the very short wavelength content, may be critical in determining response magnitude for the non-visual effects of nocturnal light.4