Cluster-size dependent behavior of pancreatic beta-cells has direct implications in islet transplantation therapy for type I diabetes treatment. Control over the cluster size enables evaluation of cluster-size-dependent function, ultimately leading to the production of beta-cell clusters with improved transplant efficacy. This work for the first time demonstrates the use of microcontact-printing-based cell patterning of discrete two- and three-dimensional clusters of pancreatic beta-cells. Both single and multiple cell layers are confined to a 2D area by attaching to patterns of covalently linked laminin and not adhering to surrounding polyethylene glycol. Cell clusters were successfully formed within 24 h for printed patterns in the range 40-120 mu m, and simple modulation of the initial cell seeding density leads to the formation of multiple cell layers. Semiquantitative fluorescence microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy were used to extensively characterize the surface chemistry. This technique offers exceptional control over cell cluster shape and size, and not only provides an effective tool to study the cluster-size-dependent behavior of pancreatic beta-cells but also has potential applicability to numerous other cell lines.