Specific homologous RIAs for the ovine neurophysins (oN-I/II, oN-III) were established; the assays had a sensitivity capable of measuring basal levels of the pituitary proteins in sheep plasma. Reduction in blood volume of ewes caused a 200-fold increase in oN-III with minimal changes in oN-I/II levels. After infusion of either hypertonic saline or nicotine, a similar specific increase in oN-III was observed, occurring at a time when the free water clearance rate decreased. Intramuscular administration of estradiol benzoate caused an increase in jugular plasma oN-I/II concentrations without affecting the oN-III concentrations. In one of six lactating ewes, there was an increase in oN-I/II during suckling. The release of oN-I/II, which was accompanied by small increases in oN-III, exhibited a pulsatile profile similar to that reported for the secretion of oxytocin under similar conditions. During vaginal distension, increases in intramammary pressure were accompanied by elevations in levels of oN-I/II and oN-III. It is concluded that oxytocin-related events are associated predominantly with the release of oN-I/II and that stimuli that induce vasopressin release also cause elevation of oN-III. These correlations are consistent with our previous conclusion from biochemical and amino acid sequence analysis of the oNs.