Choline acetyltransferase (CAT) activity of the striatum, preoptic area (POA), and pars distalis (PD) of the anterior pituitary gland was measured on each day of the 4-day estrous cycle of adult Wistar rats. Only in the PD did CAT activity vary significantly during the cycle, with the activity being lower during estrus than at other stages of the cycle. The CAT activity of the PD was extremely low, approximately 500 nmol/h/g protein, and, therefore, the specificity of the CAT activity of the PD was verified in studies on substrate requirement, the effect of a CAT inhibitor, and by paper chromatography of the reaction products. Measurement of CAT activity in saline-perfused glands, in sections of tissue taken from the lateral extremes of the PD and in hypophysial portal blood showed that the CAT activity of the PD was intrinsic, and not due to CAT activity in entrapped blood or adjacent tissues. The precise cellular origins of CAT in PD and the significance of the cyclical variation require further investigation.