The viability of erythrocyte-free malaria parasites (Plasmodium yoelii) was assessed with the cationic fluorescent dye, rhodamine 123 (R123). Parasites were freed from infected mouse erythrocytes either by saponin lysis or by Tris-ammonium chloride lysis and incubated with R123 at 37 degrees C. The parasite-associated dye was extracted with iso-butanol and measured with a fluorescent spectrophotometer. R123 accumulated intensely in free parasites prepared by saponin lysis but only weakly in those prepared by Tris-NH4Cl. Very little dye accumulated in free parasites fixed with 2% glutaraldehyde or with 3.7% formaldehyde, or in those heated at 50 degrees C for 15 min. Similar results were obtained by observations with an epifluorescent microscope. Free parasites incubated in hypotonic solutions did not accumulate the dye. Only parasites intensely accumulating R123 incorporated [3H] hypoxanthine. These results indicate that only living parasites are stained with R123, which can therefore be used to monitor the viability of erythrocyte-free malaria parasites.