Current issues in both tissue engineering and cell biology deal with cell behavior extensively in 3D. Here, we explore synchrotron radiation micro-computed tomography as a tool for morphological characterization of such 3D cellular constructs, providing micrometer resolution in soft and hard tissues. Novel image processing techniques allowed quantification of local and global cell distributions, cell density, adhesive cell culture surface, and scaffold geometry. For proof of concept, we applied this technique to characterize the morphology of two cell cultures of different phenotypes, namely human dermal fibroblasts and mouse calvarial osteoblast-like cells, both seeded on a polymer multifilament yarn. From 3D visualizations in these case studies, we saw that the fibroblasts spanned between the yarn filaments and in this way encapsulated the yarn, whereas the osteoblast-like cells lined the filament surfaces and did not span between them. Differences found in cell distribution as a function of distance to the median yarn axis and the closest filament surface, respectively, quantified these qualitative impressions gained from 3D visualizations. Moreover, the volume-normalized adhesive surface differed by one order of magnitude between the two phenotypes. Our approach allows quantitative correlation of local scaffold geometry and cell morphology. It can be used to investigate the influence of cell phenotype as well as various biochemical agents on tissue engineering constructs and the behavior of cells in culture. [on SciFinder (R)]