Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.AFM images of LEC show structures that correlate well with SEM images. Dried‐coated, dried‐uncoated and wet‐fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried‐coated, dried‐uncoated and wet‐fixed LEC. Comparison of dried‐coated (SEM) and wet‐fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high‐resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried‐coated, dried‐uncoated and wet‐fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission