The influence of photoperiod on serum prolactin levels and prolactin release induced by thyrotropin releasing hormone (TRH) was determined in ewes maintained under the following lighting regimes: Room 1, lighting mimicked natural changes in photoperiod; Room 2, annual photoperiod changes condensed into 6 mo with short days in June; Room 3, same as Room 2 except photoperiod changed abruptly from 16.5 to 8.0 h on 21 Mar. and back to 16.5 h on 21 June; Room 4, constant light. Weekly blood samples were obtained from February to August. Additionally, blood samples were collected before and after treatment with 10 μg TRH on 19 May, 13 June, 27 June and 19 July. Prolactin levels were elevated in ewes exposed to long days or constant light. The mean of all pre-TRH samples was significantly correlated with stress-induced elevations in prolactin (highest pre-TRH value) (r = 0.72) and area under the TRH-induced release curve (r = 0.56). The prolactin release in response to TRH was greatest in ewes exposed to long days or constant light. Abrupt increase of day length elevated pretreatment prolactin levels (P < 0.01) and increased area under the response curve (P < 0.05). Key words: Photoperiod, TRH, prolactin, ewes