The kinetics of initiation of in vitro RNA synthesis by Escherichia coli RNA polymerase on the chromatin template of chick oviduct was examined and compared to initiation on the template of deproteinized chick DNA. The formation of rapidly starting, highly stable binary complexes (RS complexes) between RNA polymerase and chromatin was an apparent first order process with a half-time (t1/2) of 9.4 min. By contrast, the t1/2 of formation of RS complex on chick DNA was 1.3 min. The apparent first order rate constants of RNA chain initiation from performed RS complexes were 0.57 s-1 for chromatin and 0.98 s-1 for DNA. Thus, although the formation of RS complex on chromatin was much slower than on DNA, the actual rate of RNA chain initiation on chromatin was similar to that on DNA. The effects of temperature on the formation of RS complex were examined. On chick DNA, both the rate of formation and the amount of RS complex formed decreased as the temperature of preincubation was lowered from 37 degrees to 0 degrees. On chromatin, the t1/2 of formation of RS complex was independent of temperature, and the amount of complex formed was only slightly affected at changing, temperatures. The kinetics of initiation on chromatin is consistent with the hypothesis that the rate-limiting step in the formation of RS complex is dissociation of RNA polymerase from nonspecific interaction with chromosomal proteins. Furthermore, chromosomal proteins seem to interact with initiation sites to facilitate the opening of the DNA strands required for RS complex formation.