The polyene antibiotic filipin was used as a probe for cholesterol in the nuclear membranes of cardiac muscle and capillary endothelial cells. Samples of perfusion-fixed rabbit and rat myocardium were exposed to filipin in glutaraldehyde fixative, prior to examination by standard freeze-fracture techniques. Quantitative analysis of the responses of the inner and the outer nuclear membranes of the two cell types was carried out by measuring the numerical density of filipin-induced lesions in each. Cardiac muscle cell outer nuclear membranes displayed few or no lesions (less than 0.1 micron-2), and their inner nuclear membranes were completely resistant to the treatment. By contrast, a marked response to filipin was found in the endothelial nuclear envelope, the outer membrane showing an average of 250 lesions micron-2, and the inner membrane 63 lesions micron-2. These results are interpreted as a direct reflection of differences in cholesterol content between the outer and inner nuclear membranes of the two cell types. The significance of the apparent differences in cholesterol content demonstrated between the nuclear envelopes of different cell types is discussed with reference to biochemical data, plasma membrane cholesterol content, nuclear membrane function and the supply of exogenous cholesterol.