This study determined that the effect of 9-β-d-arabinofuranosyl-adenine (adenine arabinoside, Ara-A) upon vaccinia virus plaque development in the stable monkey kidney line, LLC-MK2, was increased approximately 40-fold when an inhibitor of adenosine deaminase (ADA) was added to the tissue culture media along with infective inocula. The concentration of Ara-A required to completely suppress plaque development (total plaque inhibitory concentration100; TPIC100) was greater than 10 μg/ml. However, when ADA activity was inhibited, the TPIC100 was 0.5 μg/ml or less. Chromatographic assay of arabinosylpurines in the media provided evidence that adenine arabinoside was rapidly deaminated to 9-β-d-arabinofuranosylhypoxanthine by the cellular monolayers, in the absence of animal serum, and that the rate of deamination, at 5 μg/ml, by the cells was equal to the rate of diffusion of Ara-A across the cellular membrane. The half-life of Ara-A in the media, starting with 5 μg/ml, was 2 to 3 h and shorter at lower concentrations. The study demonstrates the profound effect that an indicator system, acting as an intact biological unit, can have upon a potential antiviral compound.