HydrolyJi,= ol'¢ndoth=lil~ l by rat kidney membran~ ~ats invcJtiilated min~ a r¢'~r,~-phz~ HPLC and an autonutted I~s.pham protein re.qa~r. F_.ndothelin I w~,= hydrolyz~;d into four major fragments which w=m d=t~:ted by HPL¢, Pho.phoramidon, an inhibitor of neutral endo~ptid~t,c 24,1 l, almost =ompl=t~y supp~_~d_ the production of thr¢~ fralrnrntr,, but on= fralr, mcnt was not affcrt~ by tl'~ inhibitor, Anallais ofN-terminal ~quence.s of the d¢l~radation products revcal~ that the pho~phoramidon-~cmlitiv¢ fralmenta were s~=rated by cl¢~vai~ at th~ Seal-Lea" bond of endoth¢lin I that wa~ ideation| with it~ clcavall~ ~it¢ by purified rat ¢ndoi~ptida~ 24,1 I, reported previously. Th= phozphoramidon.in~miti,,~ frallrnent was produc~.d by cl~val~ at ~u~=Asp '', which was distinct from the sites by endopeptida~ 24,11, but ¢orRtponded to that by a phosphoramidon.in~ndtivc m©tallo-endop~ptidar,~ recently it, elated from rat kidney mcmbran¢~ by us [(1992) Eur. J. Bioch~m, 204, 547-552]. Kinetic determination o1" endothclin I hydroly~lis by the i~lat~ eaaym¢ yielded walu~ of ,~,,=71.5 aM and ka,. = 1.49 s "~' Siv[n~ a ratio of A'.,/E,~=2,O8 x tO' tC',M", The/f~ value wutl much hisher and th¢ L'.d/~. value wa,~ much lower than tho~t £or rat ~ndo~ptida~ 24, I 1 reportad previou,,ly, Thus, endop.ptidarm 24,11 app*ar,, to h>,drolyz~ ¢ndoth~lin I more ¢fficicntly than the iml~t=d enzyme dot~. Both enzymes may play phyr, iologir.al rol~ in the rn¢taboli~m ofendothelin I by rat kidney m~ntbrun~ in vivo,