Oxytocin is involved in the regulation of reproductive and social behaviours, mood and stress responses. Previous work has indicated that oxytocin levels are regulated by circadian phase in brain tissue and plasma of both monkeys and rats, but in the cerebrospinal fluid (CSF) only of monkeys. We examined oxytocin levels in plasma and CSF of rats at two daily phases in darkness (mid subjective day and late subjective night) and after a 30 min exposure to light. We found that an apparent day-night difference in plasma oxytocin levels was eliminated by prior habituation to handling and injections. A previously reported daily oxytocin rhythm in rat plasma may instead reflect a rhythm of responsiveness to stressful experimental procedures. We also report for the first time that oxytocin levels in the CSF of rats are regulated by circadian phase and by prior exposure to light.