A reproducible plaque assay procedure for the quantitation of Venezuelan equine encephalomyelitis (VEE) virus in chick fibroblast monolayer cultures is described that is slightly more sensitive (0.20 log) than the mouse assay. The standard tissue monolayer assay was modified by using tris(hydroxylmethyl)aminomethane (Tris) as a buffer for the agar overlay. Chick fibroblast monolayer cells infected with VEE virus can be maintained under the Tris agar overlay for the time necessary for plaque formation, using a conventional laboratory incubator without a carbon dioxide atmosphere. The adsorption of the virus to chick fibroblast cells was more efficient when the virus inoculum was serially diluted in heart infusion broth at pH 6.0 and when the virus adsorbed for 4 hours at 25 C before the agar overlay was added. Statistically, the plaque and mouse assays showed a linear relationship, 1:1 ratio, between log plaque-forming units and log mouse intracerebral LD50 units; thus, the plaque assay can replace the mouse assay for determining virus concentration of VEE products.