Studies on the effect of the temperature requirement for RNA chain initiation by Escherichia coli RNA polymerase on DNA, chromatin, and reconstituted chromatin were carried out in order to better understand the nature of the initiation process. Varying the temperature or ionic strength during preincubations had little effect on the formation of stable preinitiation complexes (RS complexes) between RNA polymerase and chromatin. This observation was in marked ocntrast to similar studies performed on native DNA and indicates that initiation sites for RNA synthesis on chromatin are different from those on double-stranded DNA and resemble more closely initiation of RNA synthesis on single-stranded DNA. These results suggest that the local unwinding of the initiation region which is required for RNA chain initiation on native DNA may not be a prerequisite for RNA initiation on chromatin. The results of studies on reconstituted chromatin devoid of different classes of chromatin proteins demonstrate that both histone and nonhistone fractions are essential in maintaining the characteristics inherent to initiation of RNA synthesis on chromatin. Removal of either moderatley lysine-rich histone or arginine-rich histone fractions led to the complete loss of the characteristic "chromatin type" initiation pattern for RNA synthesis, whereas, removing lysine-rich (Fi) histone had no effect. Single strand specific and double strand specific antibodies to DNA and a single strand specific nuclease were used as probes to examine the structure of chromatin sites for initiation of RNA synthesis. Their effects on RNA synthesis suggest that initiation sites on chromatin are not located in freely accessible single-stranded or double-stranded regions of DNA.