Biological macromolecular interactions between proteins, transcription factors, DNA and other types of biomolecules, arefundamentally important to several cellular and biological processes. 3D Multi-channel confocal microscopy and colocalizationanalysis of fluorescent signals have proven to be invaluable tools for detecting such molecular interactions. The aim of thiswork was to quantify colocalization of the FOXP3 transcription factor in 3D cellular space generated from the confocal 3D imagesets. 293T cells transfected with a conditionally active form of FOXP3 were stained for nuclei with Hoechst, for FOXP3 withanti-FOXP3 conjugated to PE, and 4-hydroxytamoxifen used as protein translocation and activation agent. Since the protein signal was weak and nonspecific intensity contributions were strong, it was difficult to perform colocalization analysis and estimatecolocalization quantities. We performed 3D restoration by deconvolution method on the confocal images using experimentallymeasured point spread functions (PSFs) and subsequently a color shift correction. The deconvolution method eliminatednonspecific intensity contributions originating from PSF imposed by optical microscopy diffraction resolution limits and noisesince these factors significantly affected colocalization analysis and quantification. Visual inspection of the deconvolved 3Dimage suggested that the FOXP3 molecules are predominantly colocalized within the nuclei although the fluorescent signals fromFOXP3 molecules were also present in the cytoplasm. A close inspection of the scatter plot (colocalization map) and correlationquantities such as the Pearsons and colocalization coefficients showed that the fluorescent signals from the FOXP3 molecules and DNA are strongly correlated. In conclusion, our colocalization quantification approach confirms the preferential association ofthe FOXP3 molecules with the DNA despite the presence of fluorescent signals from the former one both in the nuclei and cytoplasm.