Abstract: The intensity of gene transcription is generally reflected by the level of RNA polymerase II (RNAPII) recruitment to the gene. However, genome-wide studies of polymerase occupancy indicate that RNAPII distribution varies among genes. In some loci more polymerases are found in the 5' region, whereas in other loci, in the 3' region of the gene. We studied the distribution of elongating RNAPII complexes at highly transcribed GAL-VPS13 locus in Saccharomyces cerevisiae and found that in the cell population the amount of polymerases gradually decreased toward the 3' end of the gene. However, the conventional chromatin immunoprecipitation assay averages the signal from the cell population, and no data on single cell level can be gathered. To study the spacing of elongating polymerases on single chromosomes, we used a sequential chromatin immunoprecipitation assay for the detection of multiple RNAPII complexes on the same DNA fragment. Our results demonstrate uniform distribution of elongating polymerases throughout all regions of the GAL-VPS13 gene.