||Almost all life on earth has adapted to natural cycles of light and dark by evolving circadian and circannual rhythms to synchronize behavioural and physiological processes with the environment. Artificial light at night (ALAN) is suspected to interfere with these rhythms. In this study we examined the influence of ALAN on nocturnal melatonin and sex steroid blood concentrations and mRNA expression of gonadotropins in the pituitary of European perch (Perca fluviatilis) and roach (Rutilus rutilus). In a rural experimental setting, fish were held in net cages in drainage channels experiencing either additional ALAN of ~15 lx at the water surface or natural light conditions at half-moon. No differences in melatonin concentrations between ALAN and natural conditions were detected. However, blood concentration of sex steroids (17β-estradiol; 11-ketotestosterone) as well as mRNA expression of gonadotropins (luteinizing hormone, follicle stimulating hormone) was reduced in both fish species. We conclude that ALAN can disturb biological rhythms in fish in urban waters. However, impacts on melatonin rhythm might have been blurred by individual differences, sampling methods and moonlight. The effect of ALAN on biomarkers of reproduction suggests a photo-labile period around the onset of gonadogenesis, including the experimental period (August). Light pollution therefore has a great potential to influence crucial life history traits with unpredictable outcome for fish population dynamics.