We developed a 96-well microplate assay to quantitate anchorage-independent growth of transformed cells. Wells of tissue culture microtiter plates are coated with poly(2-hydroxyethyl methacrylate) (poly-(HEMA)) to prevent cell attachment, and cells suspended in liquid media are seeded into the treated plates. Cell growth is assessed by tetrazolium dye reduction or by counting [3H]thymidine incorporation into DNA. There appeared to be a close correlation between growth in poly(HEMA)-coated plates and colony formation in soft agar, i.e., fibroblasts transformed by various oncogene proliferated in the coated plates, whereas their normal counterparts did not. Transformed cells on the nonadhesive surface were round and formed multicellular spheroids which resembled colonies in soft agar. Cells carrying either temperature-sensitive v-src or inducible v-Ki-ras oncogene proliferated on poly(HEMA)-coated plates only when they displayed transformed phenotype. This method is simple, quick, quantitative, and economical and in many cases might be more practical than conventional soft agar colony formation assay for measurement of anchorage-independent growth.