Abstract: A major limitation of chromatin immunoprecipitation (ChIP) lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative ChIP assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein. We achieved this by spiking every immunoprecipitation reaction with the formaldehyde cross-linked in vitro counterparts of TFIIIB-, TFIIIC- and pol III-DNA complexes, measuring the in vitro occupancies of the corresponding factors on a DNA probe and determining probe recovery by quantitative PCR. Analysis of nine pol III-transcribed genes with diverse sequence characteristics showed a very high occupancy by TFIIIB and pol III (pol III occupancy being generally ~70% of TFIIIB occupancy) and a TFIIIC occupancy that ranged between ~5% and 25%. Current data suggest that TFIIIC is released during transcription in vitro and it has been proposed that TFIIIB suffices for pol III recruitment in vivo. Our findings point to a transient nature of the TFIIIC-DNA interaction in vivo, with no significant counter-correlation between pol III and TFIIIC occupancy, and instead to a dependence of TFIIIB-DNA and TFIIIC-DNA complex maintenance in vivo on pol III function.