A purification procedure is described by which we obtained DNA-dependent RNA polymerase B (or II) from third-instar larvae of Drosophila melanogaster in essentially pure form. The enzyme is similar to the analogous polymerases from other eukaryotes in its enzymic and structural properties. It preferentially transcribes DNAs containing single-stranded regions, and it is inhibited by low amounts of the toxin alpha-amanitin; 50% inhibition occurs at an alpha-amanitin concentration of 0.03 mug/ml. Dodecylsulfate-polyacrylamide gel electrophoresis resolves the purified Drosophila polymerase B into ten polypeptides with molecular weights as follows: 1 (174000), 2 (137000), 3 (34000), 4 (22000), 5 (18000), 6 and 7 (16000), 8 (15000), and 9 and 10 (less than 15000). The relative amounts of polypeptides 1-4 were constant at molar ratios of approximately 1:1:1:2 in different preparations of the enzyme, while the amounts of polypeptides 5-10 showed more variation. An antiserum directed against the Drosophila RNA polymerase B inhibited the activity in vitro of the B enzymes from Drosophila, yeast, and calf thymus. However, only the Drosophila enzyme gave a precipitin reaction with the antiserum. When the antiserum was added to Drosophila RNA polymerase B at different stages of the purification, the resulting precipitates were found to contain nearly constant proportions of seven of the ten polypeptides present in the purified enzyme.