The enzyme kinetic parameters of the degradation of luteinizing hormone-releasing hormone (LH-RH) and L-cystine-bis-(4-nitroanilide) (Cys-NA) by rat hypothalamic (HYP) and pituitary (PIT) extracts and the effect of various oligopeptides on the rate of LH-RH inactivation were investigated in vitro. The 105,000 × g supernatant of 1 rat HYP inactivated 57 μg LH-RH during a 30 min incubation (K<sub>m</sub> = 12.4 μM,V<sub>max</sub> = 2.33 μg LH-RH/mg protein/min), and of one rat anterior PIT, 48 μg LH-RH during 30 min of incubation (K<sub>m</sub> = 12.2 μM, V<sub>max</sub> = 8.0 μg LH-RH/mg protein/min). The synthetic substrate Cys-NA competitively inhibited LH-RH degradation with a Ki of 8.5 μM in the HYP and 6 μM in the PIT enzyme preparation. Vice versa, LH-RH also competitively inhibited the cleavage of Cys-NA with inhibition constants of 14 μM (HYP) and 15 μM (PIT) indicating that the 2 substrates are probably cleaved by the same enzyme. The most effective inhibitors of LH-RH degradation were found to be angiotensin-related peptides, neurotensin, bradykinin, and bacitracin. A relatively weak effect was obtained with oxytocin, enkephalin and puromycin. It is concluded that endogenous oligopeptides such as angiotensins, neurotensin, bradykinin, etc., may possibly influence LH-RH degradation in the PIT and the HYP. The synthetic substrate Cys-NA may be an appropriate substrate for measuring the activity of an LH-RH-degrading peptidase, which therefore could be classified as arylamidase.