As the microenvironment of most cells in a multicellular organism is three dimensional, but most culture conditions are two dimensional, we evaluated the impact of 2D versus 3D culture conditions on cell morphology using the microglial cell -line, BV - 2, as a model. BV - 2 cells were either cultured on Puramatrix Peptide Hydrogel, an inert 3D matrix, or on poly-D-lysine (PDL) coated dishes (2D). In additional experiments, Puramatrix Peptide Hydrogel or PDL coated dishes were covered with the extracellular matrix proteins, fibronectin and collagen, respectively. Furthermore, BV - 2 cells were exposed to the phorbol ester, phorbol 12-myristate 13-acetate (PMA), a ramification inducing substance in microglia, under 2D or 3D conditions. Distinct cell pheotypes were quantified and statistically evaluated using C2 test. We found that 2D culture conditions promote amoeboid phenotypes, but 3D condition promotes a ramified phenotype. PMA did not show an additional affect on ramification in 3D culture conditions, in comparison to BV - 2 cells cultured on PDL. Interestingly, fibronectin suppressed PMA induced ramification in BV - 2 cells. A 3D collagen matrix not only suppressed ramification, but also inhibited the invasion of the matrix. Thus, our experiments indicate that an inert 3D matrix is sufficient to promote ramification in BV - 2 cells.