Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.