Millions of individuals habitually expose themselves to room light in the hours before bedtime, yet the effects of this behavior on melatonin signaling are not well recognized. We tested the hypothesis that exposure to room light in the late evening suppresses the onset of melatonin synthesis and shortens the duration of melatonin production. In a retrospective analysis, we compared daily melatonin profiles in individuals living in room light (<200 lux) vs. dim light (<3 lux). Healthy volunteers (n = 116, 18-30 yr) were recruited from the general population to participate in one of two studies. Participants lived in a General Clinical Research Center for at least five consecutive days. Individuals were exposed to room light or dim light in the 8 h preceding bedtime. Melatonin duration, onset and offset, suppression, and phase angle of entrainment were determined. Compared with dim light, exposure to room light before bedtime suppressed melatonin, resulting in a later melatonin onset in 99.0% of individuals and shortening melatonin duration by about 90 min. Also, exposure to room light during the usual hours of sleep suppressed melatonin by greater than 50% in most (85%) trials. These findings indicate that room light exerts a profound suppressive effect on melatonin levels and shortens the body's internal representation of night duration. Hence, chronically exposing oneself to electrical lighting in the late evening disrupts melatonin signaling and could therefore potentially impact sleep, thermoregulation, blood pressure, and glucose homeostasis.