The hypothesis of functional hemizygosity has been examined for the α‐amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, CHW, M3‐1 and CHO‐Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α‐amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO‐Kl lines became resistant to α‐amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3‐1 cell lines. The remaining activity in the latter cell lines shows α‐amanitin sensitivity similar to that seen with the wild‐type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α‐amanitin is present in CHO and CHO‐Kl cells, two copies of this gene are functional in the CHW and M3‐1 cell lines.