BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion. METHODS: We investigate the influence of cell density on homogeneity and differentiation during culture expansion of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS). MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q-PCR. RESULTS: The morphological observation and the phenotypic analyses showed that MSCs after 3 weeks cultivation constituted a phenotypically homogenous MSC cell population, which at low levels expressed genes for different cell lineages, confirming their multilineage plasticity, without actual differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which persists independent of cell density can be used for clinical therapies.