The applicability of two photon excitation 4Pi confocal fluorescence microscopy to biological imaging is demonstrated. We show that 4Pi confocal microscopy in combination with a simple deconvolution algorithm allows axial localization and quantification with 0.14 &mgr;m resolution in a biological sample. The 4Pi‐confocal microscope extends the applicability of far field fluorescence microscopy to high resolution three‐dimensional imaging and quantification of subcellular structures.