AbstractThe ribonuclease (RNase) activity associated with the surface of cells (primarily the Vero line of Green monkey kidney) was assayed under conditions where all cells remained viable as members of colonies or monolayers. RNase activity associated with individual clones was revealed as clear zones of hydrolyzed (acid‐soluble) RNA against an opaque background of acidprecipitated RNA in an isotonic agarose‐yeast RNA overlay. High resolution assays for single‐ and double‐stranded RNase were achieved by measuring the loss in activity of infectious Sindbis virus RNA, and of the antiviral state induced by poly(rI). poly(rC), respectively.Experiments using these assay procedures revealed that virtually all of the RNase activity associated with viable (intact) vertebrate cells in culture was contributed by the serum in the growth medium, and was superficially adsorbed to the cell surface, rendering it relatively easy to remove by extensive washing.A confluent monolayer of 2 × 106unwashed Vero cells contained the equivalent of 500 ng of pancreatic RNase, representing about 5% of the total activity initially present in the serum of the growth medium. In terms of serum nuclease activity, only 0.45 ng equivalents of pancreatic RNase in 500 μl were required to destroy 50% of the activity of an infectious Sindbis virus RNA preparation in 15 minutes at 37°C.Cells grown in the absence of serum and cells washed about ten times had comparably low levels of RNase activity–about 100‐fold less than unwashed cells grown in the presence of 6% calf serum.The isotonic yeast RNA:agarose overlay procedure may be useful to identify and isolate cell mutants with different endogenous levels, or binding capacities, of surface RNase activity.Operationally, these studies describe the assay of cell surface‐associated RNase activity under conditions where all cells remain viable as members of individual colonies or monolayer populations. Specifically, the assay measures the solubilization of yeast RNA in an isotonic agarose mixture overlaying the cells. We describe its use to determine how much of the total RNase activity in viable cell monolayers or colonies is contributed by serum in the growth medium. In addition, we describe the results of two biological assays used as higher resolution probes for cell surface ribonuclease activity: (i) single‐strand (ss) RNA, in the form of infectious RNA from Sindbis virus, and (ii) double‐stranded(ds)‐RNA, in the form of poly(rI). poly(rC) as an inducer of an interferon‐