Involvement of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in regulation of GnRH release was tested by determining the effect of CaM kinase II antagonists (KN-62 or KN-93) on GnRH release from rat or cattle infundibular (stalk median eminence) explants. Preincubation of male rat infundibular explants for 30 min with KN-62 (0.5, 1, 5 or 10 µM) 1.5 h prior to the addition of 59.3 mM (high) K⁺ resulted in a dose-dependent suppression of GnRH release. A longer pretreatment period (2 h) of rat infundibular explants with KN-62 (1 or 10 µM) appeared to enhance the suppressive effect of the CaM kinase II antagonist. Exposure (2 h) of rat infundibular explants to 10 µM, but not 0.1 µM KN-93, resulted in a complete inhibition of high K⁺-induced GnRH release. Exposure of steer infundibular explant halves to KN-62 (50 or 100 µM) or KN-93 (50 µM) inhibited high K⁺-induced GnRH release. Likewise, treatment of heifer infundibular explant halves with KN-93 (50 µM) abolished high K⁺-induced GnRH release. The period of exposure required for KN-62 to elicit its effect was relatively short since exposure of KN-62 (100 µM) for only 91–150 min of incubation was sufficient to block high K⁺-induced GnRH release from steer infundibular explant halves. In conclusion, these results: (1) support the hypothesis that CaM kinase II is involved in GnRH release from the rat and cattle infundibulum, (2) demonstrate that the effect of CaM kinase II on GnRH release from cattle infundibula is independent of reproductive state, (3) confirm previous reports supporting Ca²⁺ and CaM involvement in GnRH release from rat and cattle infundibula and (4) establish that infundibular explants incubated in vitro are useful for studying selected mechanisms regulating hypothalamic neurohormone release from neuron terminals.