Activity of RNA polymerases I, II and III (distinguished usingα-amanitin) and activity of DNA polymerasesαandβ(distinguished using N-ethylmaleimide) were assayed for varying intervals and at varying substrate (UTP or dTTP) concentrations in the purified nuclear fraction from corneal epithelium of carbamylcholine-treated and control eyes of rabbits with resurfacing acid burn defects. Incorporation was linear with time for all enzymes up to 30 min. In 10 min assays at varying substrate concentrations, all polymerases from carbamylcholine-treated eyes had significantly elevated Vmax compared to corresponding control enzymes. The drug also increased apparent affinity of RNA polymerase II for UTP and apparent affinity of DNA polymerases a and 3 for dTTP. Results are discussed in relation to potential mechanisms by which effects of carbamylcholine on polymerase activity may be mediated.