Infection of human erythroleukemic K562 cells by encephalomyocarditis virus readily resulted in establishment of persistently infected cultures. In contrast to the usual typical lytic infection by encephalomyocarditis virus, in which trypan blue staining of cells reaches close to 100% by about 15 h postinfection, K562 cell cultures required 3 to 4 days postinfection to reach a maximum of about 80 to 90% cell staining. The proportion of K562 cells taking up stain gradually decreased to about 10% of those present by about 13 days postinfection; during this time, virus yield per day measured by either plaque or hemagglutination titration fell about 10-fold. The decrease in percent staining was followed by waves of increased staining accompanied by increased virus production. Virus-producing cultures were maintained for over 3 months. Evolution of both virus and cells accompanied establishment of persistence in that plaque size changed from about 7 mm in diameter for the original virus to less than 1.5 mm by day 20 postinfection and most of the cells cloned from persistently infected cultures were resistant to superinfection with the original virus. Resistance was due, at least in part, to reduced virus attachment in that binding of 3H-labeled virus to cloned resistant cells was about 2% of that to uninfected cells.