One important function of endocannabinoids and related lipid mediators in mammalian central nervous system is modulation of pain. Evidence obtained during the last decade shows that altered levels of these compounds in the brain accompany decreases in pain sensitivity. Such changes, if sexually dimorphic, could account for sex differences in pain and differences that occur during different phases of the hormonal cycle in females. To examine this possibility, we measured the levels of the pain-modulatory lipids anandamide, 2-arachidonoyl glycerol, N-arachidonoyl glycine, N-arachidonoyl gamma amino butyric acid, and N-arachidonoyl dopamine in seven different brain areas (pituitary, hypothalamus, thalamus, striatum, midbrain, hippocampus, and cerebellum) in male rats, and in female rats at five different points in the estrous cycle. The cerebellum did not demonstrate a change in endocannabinoid production across the estrous cycle, whereas all other areas tested showed significant differences in at least one of the compounds measured. These changes in levels occurred predominantly within the 36-h time period surrounding ovulation and behavioral estrus. Differences between males and females were measured as either estrous cycle-independent (all estrous cycles combined) or cycle-dependent (comparisons of males to each estrous cycle). In cycle-independent analyses, small sex differences were observed in the pituitary, hypothalamus, cerebellum, and striatum, whereas no differences were observed in the thalamus, midbrain, and hippocampus. In cycle-dependent analyses, the hypothalamus and pituitary showed largest sex differences followed by the striatum, midbrain, and hippocampus, whereas no sex differences were measured in thalamus and cerebellum. These data provide a basis for investigations into how differences in sex and hormonal status play a role in mechanisms regulating endocannabinoid production and pain.