Spermine, at concentrations similar to those normally present in the cytosol of liver cells, facilitates the transport of phosphate into mitochondria and thus its accumulation within the matrix space. Both mersalyl and N-ethylmaleimide (NEM) inhibit phosphate influx either in the absence or in the presence of spermine. These inhibitors also inhibit, but only partially, the efflux from mitochondria of phosphate generated within the matrix space by the hydrolysis of ATP induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or the valinomycin-K+ system. The inhibition of phosphate efflux by both mersalyl and NEM is almost completely removed, unlike that of phosphate influx, by spermine. The possibility that spermine may induce phosphate efflux by damaging mitochondrial membranes and consequently inducing an unspecific permeability to phosphate is excluded by the full restoration of transmembrane potential once FCCP has been removed by albumin. Since spermine does not react with either thiol groups or thiol group reagents, the simplest explanation of the reported results is that the pathway of phosphate efflux is distinct from that of phosphate influx.