Hen oviduct RNA polymerase II and Escherichia coli RNA polymerase holoenzyme and core enzyme were used to study the initiation of RNA synthesis on chromatin. In either the presence or absence of estrogenic stimulation, changes in the level of oviduct chromatin initiation sites as measured in the presence of either homologous or heterologous polymerases followed a similar pattern. Comparison of the initiation sited utilized by these enzymes on chick oviduct chromatin indicated that these enzymes compete with each other for the same initiation regions. In contrast to chromatin, however, the majority of the initiation sites on DNA which are utilized by the oviduct RNA polymerase II are different from those utilized by E. coli holoenzyem. These results suggest that chromatin proteins are involved in the selection of initiation sites on chromatin for RNA polymerases. The in vitro transcripts of these RNA polymerases on stimulated chick oviduct chromatin were analyzed by hybridization to a cDNA probe transcribed from ovalbumin mRNA. The relative concentration of ovalbumin sequences transcribed by these three polymerases was 4:1.5:1 for oviduct RNA polymerase II, E. coli core enzyme, and holoenzyme respectively. Therefore, the efficiency of transcribing a specific gene appears to depend on the interaction between RNA polymerase and chromosomal elements in the initiation region.