Fluorescence imaging of cells is a powerful tool for exploring the dynamics of organelles, proteins, and viruses. Fluorescent adenoviruses are a model system for cargo transport from the cell surface to the nucleus. Here, we describe a procedure to quantitate adenovirus-associated fluorescence in different subcellular regions. CCD camera-captured fluorescence sections across entire cells were deblurred by a fast Fourier transformation, the background was subtracted images merged, and virus fluorescence quantitated. The validity of the deblurring routine was verified by confocal laser scanning microscopy, demonstrating that objects were neither generated nor deleted. Instead, the homogeneity of both the average intensity and the size of fluorescent particles was increased, facilitating automated quantification. We found that nuclear fluorescence of wt adenovirus, but not of a virus mutant ts1, which fails to escape from endosomes, was maximal at 90 min postinfection (p.i.). Surprisingly, nuclear fluorescence decreased at 120 min, but increased again at 240 min p.i., suggesting that wt virus targeting to the nucleus may be multiphasic and regulated. Interestingly, only the first nuclear transport period of wt but not ts1 virus coincided with a significant increase of the peripheral and decrease of the cytoplasmic regions, indicative of signal-dependent cell contraction.