Endothelial cells (EC) form three-dimensional aggregates without any scaffold, when they are exposed to microgravity (µg) simulated by a random positioning machine (RPM), but not under static 1g conditions. Here we describe a delayed type of formation of 3D structures of EC, which was initiated, when EC cultured on a Desktop-RPM remained adherent for the first five days, but spread over neighboring adherent cells forming little colonies. After two weeks, tube-like structures (TS) became visible in these cultures. They included a lumen and elongated during another two weeks of culturing. The walls of these TS consisted mainly of single-layered EC, which had produced significantly more 1-integrin, laminin, fibronectin, and alpha-tubulin than EC simultaneously grown adhering to the culture dishes under µg or under normal gravity. The amount of actin protein was similar in EC incorporated in TS and in EC growing at 1g. Interestingly, the ratio of TIMP-1 to MMP-2 found in the supernatants was lower at the seventh than at the 28th day of culturing. These results suggest that culturing EC under conditions of modeled gravitational unloading represents a new technique to study the formation of tubes which resemble vascular intimas.