The concept of fractal geometry provides an elegant tool for the quantitative and objective structural description of various objects, the fractal analysis. Fractal analysis quantifies the structural complexity of objects by a characteristic singular value, the fractal dimension (FD). It can be estimated, e.g. by the box-counting method and provides a highly integrated measure in the range 1 < FD < 2 for curves extending within a plane. In this study, fractal analysis is used for the first time to evaluate the complexity of the tight junction network between adjoining cells. Bovine brain endothelial cells were cultured under various experimental conditions and the tight junctions were drawn to scale as visualized by the freeze fracture technique. These drawings were analyzed by fractal analysis, and by two other methods commonly used in this field, viz. the strand counting (SC) and complexity index (CI) methods. In contrast to the latter methods, the FD shows no directional preference and therefore no assumptions on the dynamic properties of the network's complexity are required. Thus, FD is demonstrated to provide the most sensitive, reliable and complete measure of tight junction complexity. In combination with SC and CI, additional information can be achieved concerning the directionality of the altered arrangement of tight junctional strands. Our analysis allows for the following conclusions. (1) Defined experimental influences can modify the complexity of tight junctions that are formed between endothelial cells in vitro, and (2) these structural modifications of the tight junctions are mainly due to an altered strand branching pattern.