Isolated heart mitochondria retain matrix K+ in a K+-free medium and exchange matrix 42K+ with external K+ only slowly. This low permeability to K+ is maintained when the proton motive force is dissipated by addition of an uncoupler, but can be increased markedly in uncoupled mitochondria when (a) NADPH becomes oxidized and (b) a Ca2+ chelator or ruthenium red is added (Jung, D. W., and Brierley , G. P. (1981) J. Biol. Chem. 256, 10490-10496). This latter requirement suggests that decreased Ca2+ binding or alteration of the Ca2+ uniporter may be involved in the induction of permeability to K+ in these mitochondria. The present studies establish that La3+ (k0.5 = 1.8 nmol X mg-1 of protein) also induces K+ permeability in uncoupled mitochondria in which NADPH has been oxidized. The amount of net K+ loss or passive 42K+/K+ exchange induced by La3+ corresponds to that produced by ruthenium red or EGTA and appears to vary from preparation to preparation as a function of the endogenous adenine nucleotide (AN) content of the mitochondria. The permeability to K+ induced by all three reagents is increased by depletion of endogenous AN with PPi and strongly inhibited by low levels of exogenous AN. The optimum passive permeability to K+ develops at pH 7.5, is inhibited by Nupercaine , quinine, and dicyclohexylcarbodiimide, and is increased in a sucrose, as opposed to a KCl medium. The increased permeability to K+ appears to result from the opening of one or more K+-conducting uniport pathways, rather than K+/H+ exchange. Since Ca2+ efflux remains sensitive to ruthenium red when K+ efflux is induced, it seems unlikely that the Ca2+ uniporter itself can provide a pathway for K+ flux. The presence of such latent pathways for passive K+ permeability must be considered when defining the properties of the putative K+/H+ antiporter and during isolation and reconstitution protocols involving mitochondrial K+ transport components.