The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA–protein interactions and chromatin structure in vivo and in vitro.