The facilitation of peptide secretion from the neurohypophysis induced by increasing stimulation frequency is accompanied by action potential (AP) prolongation. One hypothesis argues that inactivation of potassium channels in the neural lobe terminal membranes, under these conditions, is the underlying mechanism which leads to AP prolongation, and, therefore, increased calcium entry and secretion per AP. Therefore, factors which are known to cause AP prolongation, such as stimulus frequency and potassium channel blocking agents, were studied and compared with regard to their ability to augment electrically evoked release of oxytocin (OT) and vasopressin (VP) from isolated rat neurointermediate lobes (s). OT release (to a constant applied stimulus of 600 spikes) was maximally facilitated by increasing frequency up to a rate of 30 Hz, whereas VP release in the same stimulus paradigm was maximal between 12 and 20 Hz. Tetraethylammonium (TEA), 4-aminopyridine (4AP) and barium each caused a significant augmentation of AP-dependent, electrically stimulated hormone release, without affecting basal levels. The magnitude of the effect of the K channel blocking agents was inversely related to the frequency of the applied stimulus. Application of either 4AP or TEA caused a shift in the range of frequency dependence for OT such that maximal release was seen at a stimulus frequency of 12 Hz, but there was no comparable change in the pattern of VP release. The maximal effects of TEA and 4AP were additive indicating that the terminals have two types of K channels which appear to be involved in the regulation of secretion. Addition of the three agents together produced maximal release at a stimulus frequency of 4 Hz, which was not facilitated further by the increase of stimulus frequency to 20 Hz. These data demonstrate the importance of potassium channels in the regulation of VP and OT secretion, and provide indirect support for the spike prolongation hypothesis of frequency facilitated secretion in the neural lobe.