Abstract Background A stable and systematic daily change in light levels at dawn and dusk provides the most reliable indicator of the phase of the day. It is likely that organisms have evolved mechanisms to use these twilight transitions as the primary zeitgeber to adjust their circadian phases. In this study, we investigated under natural illumination conditions the effects of daylight exposure restricted to twilights on the timing of testicular regression and locomotor activity of the house sparrow ( Passer domesticus ), which possesses a strongly self-sustaining circadian system. Methods and results Two experiments were performed on adult male house sparrows. Beginning in the third week of April, the first experiment examined whether exposure to natural daylight only during twilights influenced the timing of testicular regression and concomitant changes in testosterone-dependent beak color of reproductively mature sparrows. Interestingly, there was a significant delay in testicular regression and depigmentation of the beak in sparrows exposed to natural daylight (NDL) only during twilights as compared to those exposed to NDL all day. The second experiment examined twice in the year, around the equinoxes (March and September), the effects of exposure to twilights only on the daily activity rhythm of sparrows kept in an outdoor aviary. Five of 7 birds continued exhibiting entrained activity rhythms when exposed only to twilights (NDL minus day light from sunrise to sunset) in September, but not in March. Both in NDL and twilight conditions, March birds had significantly lower activity counts than September birds. Conclusion Exposure to natural daylight only during twilights delayed the timing of testicular regression and concomitant depigmentation of the beak but did not affect the daily activity rhythm in male sparrows. This suggests that daily twilights can serve as cues for regulation of the circadian activity rhythm but not for the photoperiodic regulation of testicular cycle in the house sparrow.