A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 microns inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.