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A specific enhancer for the m+ variant of echovirus 6 was isolated from uninfected, permissive host cells. The enhancer transferred the susceptibility to virus infection from permissive cells to less susceptible cells. Enhancer activity in cultured, human cells (WISH) was released by these cells into extracellular fluids at a linear rate. Maximal enhancer activity was recovered from cell monolayers that were extracted with buffered salt solutions (pH 6.6) for 5 h at 37 C. Crude enhancer preparations contained substances that reduced virus titers in permissive host cells and suppressed some of the enhancer activity. Viral inhibitory activity was removed from cellular extracts by either differential centrifugation or ion exchange chromatography. The enhancer, in contrast to inhibitory substances, remained in supernatant fractions after centrifugation at 110,000 × gfor 4 h and adsorbed to diethylaminoethyl-Sephadex columns. Enhancer preparations that were devoid of inhibitory activity increased the virus titers in less susceptible simian cell cultures (approximately 100-fold) to the titers attained by fully permissive human cell cultures. A direct relationship between protein concentration and enhancer activity in cellular extracts was demonstrated. Partial purification of the enhancer by differential centrifugation and ammonium sulfate precipitation increased the specific activity of enhancer (units/mg of protein) by 13-fold.