Electromagnetic focusing theory of light predicts that at high apertures field components arise that are polarized perpendicular to the initial polarization. Although vectorial depolarization has received considerable attention in focusing theory, no evidence has been presented as to its relevance in experiments. We measure the intensity of the perpendicularly orientated field in the focal region by utilizing monomolecular, fluorescent polydiacetylene layers whose transition dipoles are orientated in a single direction. For a 1.4 numerical aperture oil objective lens illuminated with linearly x-polarized light, we find that the integral of the modulus squared of the y-polarized focal field amounts to 1.5% of its x-polarized counterpart. In particular, we show here that the depolarization increases when using annular apertures. Annuli formed by a central obstruction with a diameter of 89% of that of the entrance pupil raise the integral to 5.5%. This compares well with the value of 5.8% predicted by electromagnetic focusing theory; however, the depolarization is also due to imperfections connected with focusing by refraction. Besides fluorescence microscopy and single molecule spectroscopy, the measured intensity of the depolarized component in the focal plane is relevant to all forms of light spectroscopy combining strong focusing with polarization analysis.