There is evidence that the cholinergic system modulates, in a circadian and asymmetric way, the neural activity involved in the regulation of ovulation. In the present study we measured choline acetyltransferase (ChAT) activity during the oestrous cycle in both sides of the preoptic-anterior hypothalamic area (POA-AHA) of the rat. The right side of POA-AHA showed significant changes in ChAT activity during the oestrous cycle. The activity of the enzyme was higher in the right side on the day of oestrous (47.3 ± 3.2 nmol mg−1 protein 1 h−1vs 25.8 ± 2.4, p < 0.05), whilst on the second day of dioestrous the activity was higher in the left one (30.6 ± 3.4 vs 20.0 ± 1.5). The differences in the activity of ChAT observed support the idea of the existence of asymmetry in the POA-AHA cholinergic system which varies during the oestrous cycle.